Synthesis of cyclosporin analogs

ABSTRACT

The invention is directed to isomeric mixtures of cyclosporine analogues that are structurally similar to cyclosporine A. The mixtures possess enhanced efficacy and reduced toxicity over the individual isomers and over naturally occurring and other presently known cyclosporines and cyclosporine derivatives. Embodiments of the present invention are directed toward cis and trans-isomers of cyclosporin A analogs referred to as ISA TX 247, and derivatives thereof. ISA TX 247 isomers and alkylated, arylated, and deuterated derivatives are synthesized by stereoselective pathways where the particular conditions of a reaction determine the degree of stereoselectivity. Stereoselective pathways may utilize a Wittig reaction, or an organometallic reagent comprising inorganic elements such as boron, silicon, titanium, and lithium. The ratio of isomers in a mixture may range from about 10 to 90 percent by weight of the (E)-isomer to about 90 to 10 percent by weight of the (Z)-isomer, based on the total weight of the mixture.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application Ser. Nos.60/346,201 filed Oct. 19, 2001 and 60/370,596 filed Apr. 5, 2002. Theentire disclosure of each of these applications is incorporated hereinby reference in its entirety.

FIELD OF THE INVENTION

The invention is directed to isomeric mixtures of cyclosporin analoguesthat are related to cyclosporine A. It is contemplated that the mixturespossess enhanced efficacy and/or reduced toxicity over the individualisomers and over naturally occurring and other presently knowncyclosporines and cyclosporine derivatives. In addition, the presentinvention relates to synthetic pathways for producing isomers ofcyclosporin A analogs, where such pathways vary in the degree ofstereoselectivity depending on the specific reaction conditions.

REFERENCES

The following references are related hereto or referred to herein bypatent or application number or in parenthesis by author and year at therelevant portions of this specification:

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T. Chang, L. Z. Benet, M. F. Hebert, “The effect of water-solublevitamin E on cyclosporine pharmacokinetics in healthy volunteers,” Clin.Pharmacol. Ther., Vol. 59, pp. 297–303 (1996).

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M. K. Eberle, F. Nuninger, “Synthesis of the main metabolite (OL-17) ofcyclosporin A,” J. Org. Chem., Vol. 57, pp. 2689–2691 (1992).

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D. S. Fruman, C. B. Klee, B. E. Bierer, S. J. Burakoff, “Calcineurinphosphatase activity in T lymphocytes is inhibited by FK506 andcyclosporin A,” Proc. Natl. Acad. Sci. USA, Vol. 89, pp. 3686–90 (1992).

A. Granelli-Piperno, L. Andrus, R. M. Steinman, “Lymphokine andnonlymphokine mRNA levels in stimulated human cells: kinetics, mitogenrequirements, and effects of cyclosporin A,” J. Exp. Med., Vol. 163, p.922 (1986).

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M. F. Hebert, J. P. Roberts, T. Prueksaritanont, L. Z. Benet,“Bioavailability of cyclosporin with concomitant rifampin administrationis markedly less than predicted by hepatic enzyme induction,” Clin.Pharmacol. Ther., Vol. 52, pp. 453–7 (1992).

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I. Sketris, R. Yatscoff, P. Keown, D. M. Canafax, M. R. First, D. W.Holt, T. J. Schroeder, M. Wright, “Optimizing the use of cyclosporine inrenal transplantation,” Clin. Biochem., Vol. 28, pp. 195–211 (1995).

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U.S. Pat. No. 4,108,985.

U.S. Pat. No. 4,160,452.

U.S. Pat. No. 4,210,581.

U.S. Pat. No. 4,220,641.

U.S. Pat. No. 4,256,108.

U.S. Pat. No. 4,265,874.

U.S. Pat. No. 4,288,431.

U.S. Pat. No. 4,384,996.

U.S. Pat. No. 4,396,542.

U.S. Pat. No. 4,554,351.

U.S. Pat. No. 4,771,122.

U.S. Pat. No. 5,284,826.

U.S. Pat. No. 5,525,590.

European Patent Publication No. 0 034 567.

European Patent Publication No. 0 056 782.

International Patent Publication No. WO 86/02080.

International Patent Publication No. WO 99/18120.

The disclosure of each of the above-referenced patents, patentapplications and publications is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Cyclosporine derivatives compose a class of cyclic polypeptides,consisting of eleven amino acids, that are produced as secondarymetabolites by the fungus species Tolypocladium inflatum Gams. They havebeen observed to reversibly inhibit immunocompetent lymphocytes,particularly T-lymphocytes, in the G₀ or G₁ phase of the cell cycle.Cyclosporine derivatives have also been observed to reversibly inhibitthe production and release of lymphokines (Granelli-Piperno et al.,1986). Although a number of cyclosporine derivatives are known,cyclosporine A is the most widely used. The suppressive effects ofcyclosporine A are related to the inhibition of T-cell mediatedactivation events. This suppression is accomplished by the binding ofcyclosporine to the ubiquitous intracellular protein, cyclophilin. Thiscomplex, in turn, inhibits the calcium- and calmodulin-dependentserine-threonine phosphatase activity of the enzyme calcineurin.Inhibition of calcineurin prevents the activation of transcriptionfactors such as NFAT_(p/c) and NF-κB, which are necessary for theinduction of the cytokine genes (IL-2, IFN-γ, IL-4, and GM-CSF) duringT-cell activation. Cyclosporine also inhibits lymphokine production byT-helper cells in vitro and arrests the development of mature CD8 andCD4 cells in the thymus (Granelli-Piperno et al., 1986). Other in vitroproperties of cyclosporine include the inhibition of IL-2 producingT-lymphocytes and cytotoxic T-lymphocytes, inhibition of IL-2 releasedby activated T-cells, inhibition of resting T-lymphocytes in response toalloantigen and exogenous lymphokine, inhibition of IL-1 production, andinhibition of mitogen activation of IL-2 producing T-lymphocytes(Granelli-Piperno et al., 1986).

Cyclosporine is a potent immunosuppressive agent that has beendemonstrated to suppress humoral immunity and cell-mediated immunereactions such as allograft rejection, delayed hypersensitivity,experimental allergic encephalomyelitis , Freund's adjuvant arthritisand graft vs. host disease. It is used for the prophylaxis of organrejection subsequent to organ transplantation; for treatment ofrheumatoid arthritis; for the treatment of psoriasis; and for thetreatment of other autoimmune diseases, including type I diabetes,Crohn's disease, lupus, and the like.

Since the original discovery of cyclosporin, a wide variety of naturallyoccurring cyclosporins have been isolated and identified and manyfurther non-natural cyclosporins have been prepared by total- orsemi-synthetic means or by the application of modified culturetechniques. The class comprised by the cyclosporins is thus nowsubstantial and includes, for example, the naturally occurringcyclosporins A through Z [c.f. Traber et al. (1977); Traber et al.(1982); Kobel et al. (1982); and von Wartburg et al. (1986)], as well asvarious non-natural cyclosporin derivatives and artificial or syntheticcyclosporins including the dihydro- and iso-cyclosporins; derivatizedcyclosporins (e.g., in which the 3′-O-atom of the -MeBmt-residue isacylated or a further substituent is introduced at the α-carbon atom ofthe sarcosyl residue at the 3-position); cyclosporins in which the-MeBmt-residue is present in isomeric form (e.g., in which theconfiguration across positions 6′ and 7′ of the -MeBmt-residue is cisrather than trans); and cyclosporins wherein variant amino acids areincorporated at specific positions within the peptide sequenceemploying, e.g., the total synthetic method for the production ofcyclosporins developed by R. Wenger—see e.g. Traber et al. (1977),Traber et al. (1982) and Kobel et al. (1982); U.S. Pat. Nos. 4,108,985,4,210,581, 4,220,641, 4,288,431, 4,554,351 and 4,396,542; EuropeanPatent Publications Nos. 0 034 567 and 0 056 782; International PatentPublication No. WO 86/02080; Wenger (1983); Wenger (1985); and Wenger(1986). Cyclosporin A analogues containing modified amino acids in the1-position are reported by Rich et al. (1986). Immunosuppressive,anti-inflammatory, and anti-parasitic cyclosporin A analogues aredescribed in U.S. Pat. Nos. 4,384,996; 4,771,122; 5,284,826; and5,525,590, all assigned to Sandoz. Additional cyclosporin analogues aredisclosed in WO 99/18120, assigned to Isotechnika. The termsCiclosporin, ciclosporin, cyclosporine, and Cyclosporine areinterchangeable and refer to cyclosporin.

There are numerous adverse effects associated with cyclosporine Atherapy, including nephrotoxicity, hepatotoxicity, cataractogenesis,hirsutism, parathesis, and gingival hyperplasia to name a few (Sketriset al., 1995). Of these, nephrotoxicity is one of the more serious,dose-related adverse effects resulting from cyclosporine Aadministration. Immediate-release cyclosporine A drug products (e.g.,Neoral® and Sandimmune®) can cause nephrotoxicities and other toxic sideeffects due to their rapid release and the absorption of high bloodconcentrations of the drug. It is postulated that the peakconcentrations of the drug are associated with the side effects(Bennett, 1998). The exact mechanism by which cyclosporine A causesrenal injury is not known; however, it is proposed that an increase inthe levels of vasoconstrictive substances in the kidney leads to thevasoconstriction of the afferent glomerular arterioles. This can resultin renal ischemia, a decrease in glomerular filtration rate and, overthe long term, interstitial fibrosis. When the dose is reduced oranother immunosuppressive agent is substituted, renal function improves(Valantine and Schroeder, 1995).

Accordingly, there is a need for immunosuppressive agents which areeffective and have reduced toxicity.

Cyclosporin analogs containing modified amino acids in the 1-positionare disclosed in WO 99/18120, which is assigned to the assignee of thepresent application, and incorporated herein in its entirety. Alsoassigned to the present assignee is U.S. Provisional Patent ApplicationNo. 60/346,201, in which applicants disclosed a particularly preferredcyclosporin A analog referred to as “ISA_(TX)247.” This analog isstructurally identical to cyclosporin A except for modification at the1-amino acid residue. Applicants discovered that certain mixtures of cisand trans isomers of ISA_(TX)247 exhibited a combination of enhancedpotency, and/or reduced toxicity over the naturally occurring andpresently known cyclosporins. Certain alkylated, arylated, anddeuterated derivatives of ISA_(TX)247 were also disclosed.

Typically, the disclosed mixtures in U.S. Provisional Patent ApplicationNo. 60/346,201 range from about 10 to 90 percent by weight of thetrans-isomer and about 90 to 10 percent by weight of the cis-isomer; inanother embodiment, the mixture contains about 15 to 85 percent byweight of the trans-isomer and about 85 to 15 percent of the cis-isomer;in another embodiment, the mixture contains about 25 to 75 percent byweight of the trans-isomer and about 75 to 25 percent by weight of thecis-isomer; in another embodiment, the mixture contains about 35 to 65percent by weight of the trans-isomer and about 65 to 35 percent byweight of the cis-isomer; in another embodiment, the mixture containsabout 45 to 55 percent by weight of the trans-isomer and about 55 to 45percent of the cis-isomer. In another embodiment, the isomeric mixtureis an ISA_(TX)247 mixture which comprises about 45 to 50 percent byweight of the trans-isomer and about 50 to 55 percent by weight of thecis-isomer. These percentages by weight are based on the total weight ofthe composition. In other words, a mixture might contain 65 percent byweight of the (E)-isomer and 35 percent by weight of the (Z)-isomer, orvice versa. In an alternate nomenclature, the cis-isomer may also bedescribed as a (Z)-isomer, and the trans-isomer could also be called an(E)-isomer.

Accordingly, there is a need in the art for methods of preparation ofcyclosporin analogs, including isomers of ISA_(TX)247. Syntheticpathways are needed that produce enriched compositions of the individualisomers, as well mixtures of the isomers having a desired ratio of thetwo isomers. Methods of preparation of derivatives of ISA_(TX)247 areneeded as well.

SUMMARY OF THE INVENTION

Cyclosporine and its analogs are members of a class of cyclicpolypeptides having potent immunosuppressant activity. Despite theadvantages these drugs offer with respect to their immunosuppressive,anti-inflammatory, and anti-parasitic activities, there are numerousadverse effects associated with cyclosporine A therapy that includenephrotoxicity and hepatotoxicity. Accordingly, there is a need for newimmunosuppressive agents that are as pharmacologically active as thenaturally occurring compound cyclosporin A, but without the associatedtoxic side effects.

Embodiments of the present invention provide certain mixtures of cis andtrans-isomers of cyclosporin A analogs, which are pharmaceuticallyuseful. A preferred analog is referred to as ISA_(TX)247. Mixtures ofISA_(TX)247 isomers exhibit a combination of enhanced potency andreduced toxicity over the naturally occurring and presently knowncyclosporins.

The present invention is based in part on the discovery that certainisomeric mixtures of analogues of cyclosporine provide superiorimmunosuppressive effects without the adverse effects associated withcyclosporine A. In particular, we have unexpectedly found that isomericmixtures (i.e., mixtures of both cis- and trans-isomers) ranging fromabout 10:90 to about 90:10 (trans- to cis-) of cyclosporine analoguesmodified at the 1-amino acid residue provide superior efficacy andsafety. Examples of such analogues are disclosed in WO 99/18120, andinclude deuterated and non-deuterated compounds. In particular, mixturesin the range of about 45:55 to about 50:50 (trans- to cis-) and in therange of about 50% to about 55% trans- and about 45% to about 50% cis-are found to be particularly efficacious. Moreover, it has beendemonstrated that these isomer mixtures exhibit a combination ofsuperior potency and reduced toxicity over naturally occurring and otherpresently known cyclosporines and cyclosporine derivatives.

A particularly preferred analogue (referred to herein as “ISA_(TX)247”)is structurally similar to cyclosporine A except for a modifiedfunctional group on the periphery of the molecule, at the 1-amino acidresidue. The structure of this particular isomeric analogue mixturecompared to the structure of cyclosporine A is shown in FIGS. 1A, 1B,2A, 2B.

The isomeric mixtures can be used, among other things, forimmunosuppression, and the care of various immune disorders, diseasesand conditions, including the prevention, control, alleviation andtreatment thereof.

According to embodiments of the present invention, ISA_(TX)247 isomers(and derivatives thereof) are synthesized by stereoselective pathwaysthat may vary in their degree of selectivity. Stereoselective pathwaysproduce compositions that are enriched in either of the (E) and(Z)-isomers, and these compositions may be combined such that theresulting mixture has a desired ratio of the two isomers. Alternatively,the reactions conditions of a stereoselective pathway may be tailored toproduce the desired ratio directly in a prepared mixture. The percentageof one isomer or another in a mixture can be verified using nuclearmagnetic resonance spectroscopy (NMR) or other techniques well known inthe art.

Each of the pathways typically proceeds with the application of aprotecting group to a sensitive alcohol functional group. In oneembodiment the alcohol is protected as an acetate; in other embodimentsthe protecting groups are benzoate esters or silyl ethers. Althoughacetate protecting groups are common in the art, it is important toemphasize that in many of the exemplary embodiments described hereincertain undesirable side-reactions involving an acetate protecting groupmay be avoided through the use of protecting groups such as benzoateesters or silyl ethers.

The protected compound may then serve as a precursor for a variety ofstereoselective synthetic pathways including some that utilizephosphorus-containing reagents as participants in a Wittig reaction, andinorganic elements as members of organometallic reagents. The lattertype may proceed through six-membered ring transition states wheresteric hindrance dictates the configurational outcome. Manyorganometallic reagents are available, including those that featureinorganic elements such as boron, silicon, titanium, lithium, andsulfur. Individual isomers may be prepared from single or multipleprecursors.

The ratio of the (E) to (Z)-isomers in any mixture, whether producedstereoselectively or non-stereoselectively, may take on a broad range ofvalues. For example, the mixture may comprise from about 10 to 90percent of the (E)-isomer to about 90 to 10 percent of the (Z)-isomer.In other embodiments, the mixture may contain from about 15 to 85percent by weight of the (E)-isomer and about 85 to 15 percent of the(Z)-isomer; in another embodiment, the mixture contains about 25 to 75percent by weight of the (E)-isomer and about 75 to 25 percent by weightof the (Z)-isomer; in another embodiment, the mixture contains about 35to 65 percent by weight of the (E)-isomer and about 65 to 35 percent byweight of the (Z)-isomer; in another embodiment, the mixture containsabout 45 to 55 percent by weight of the (E)-isomer and about 55 to 45percent of the (Z)-isomer. In another embodiment, the isomeric mixtureis an ISA_(TX)247 mixture which comprises about 45 to 50 percent byweight of the (E)-isomer and about 50 to 55 percent by weight of the(Z)-isomer. These percentages by weight are based on the total weight ofthe composition, and it will be understood that the sum of the weightpercent of the (E)-isomer and the (Z)-isomer is 100 weight percent. Inother words, a mixture might contain 65 percent by weight of the(E)-isomer and 35 percent by weight of the (Z)-isomer, or vice versa.

Accordingly, in one aspect, the invention provides methods of preparingan isomeric mixture of cyclosporin A analogs modified at the 1-aminoacid residue, wherein the synthetic pathway comprises the steps of:heating an acetyl-η-halocyclosporin A with triakylphosphine,triarylphosphine (e.g. triphenylphosphine), arylalkylphosphine, andtriarylarsine to produce an intermediate phosphonium halide of acetylcyclosporin A; preparing a mixture of (E) and (Z)-isomers ofacetyl-1,3-diene by stirring the intermediate phosphonium halide ofacetyl cyclosporin A with formaldehyde, optionally in the presence of alithium halide; and preparing a mixture of (E) and (Z)-isomers ofISA_(TX)247 by treating the mixture of (E) and (Z)-isomers ofacetyl-1,3-diene with a base.

In another aspect, the invention is directed to methods of preparing anisomeric mixture of cyclosporin A analogs modified at the 1-amino acidresidue, wherein the synthetic pathway comprises the steps of:converting an intermediate, e.g., protected trimethylsilyl (TMS)cyclosporin A aldehyde or acetyl cyclosporin A aldehyde to a mixture of(E) and (Z)-isomers of acetyl-1,3-diene by reacting the intermediatewith a phosphorus ylide via a Wittig reaction, optionally in thepresence of a lithium halide; and preparing a mixture of (E) and(Z)-isomers of ISA_(TX)247 by treating the mixture of (E) and(Z)-isomers of acetyl-1,3-diene with a base in the case ofacetyl-protecting group or, e.g., an acid in case of a TMS-protectinggroup.

In a further aspect, the invention is directed to methods of producingan E-isomer enriched mixture of cyclosporin A analogs modified at the1-amino acid residue, wherein the stereoselective synthesis of theE-isomer enriched material comprises the steps of: reacting an acetylcyclosporin A aldehyde with a reagent selected from the group consistingof trialkylsilylallyl boronate ester and E-γ-(trialkylsilylallyl)dialkylborane to form a β-trialkylsilyl alcohol; treating theβ-trialkylsilyl alcohol with acid to form acetyl-(E)-1,3-diene; andtreating the acetyl-(E)-1,3-diene with base to form the (E)-isomer ofISA_(TX)247.

In yet a further aspect, the invention is directed to methods ofproducing a Z-isomer enriched mixture of cyclosporin A analogs modifiedat the 1-amino acid residue, wherein the stereoselective synthesis ofthe Z-isomer enriched material comprises the steps of: reacting anacetyl cyclosporin A aldehyde with a reagent selected from the groupconsisting of trialkylsilylallyl boronate ester and(E-γ-trialkylsilylallyl) dialkylborane to form a β-trialkylsilylalcohol; treating the β-trialkylsilyl alcohol with base to formacetyl-(Z)-1,3-diene; and treating the acetyl-(Z)-1,3-diene with base toform the (Z)-isomer of ISA_(TX)247.

In a still further aspect, the invention is directed to methods ofproducing an E-isomer enriched mixture of cyclosporin A analogs modifiedat the 1-amino acid residue, wherein the stereoselective synthesis ofthe E-isomer enriched material comprises the steps of: reacting anacetyl cyclosporin A aldehyde with a lithiated allyldiphenylphosphineoxide to form acetyl-(E)-1,3-diene; and treating theacetyl-(E)-1,3-diene with base to form the (E)-isomer of ISA_(TX)247.

In yet a further still aspect, the invention provides method ofproducing a Z-isomer enriched mixture of cyclosporin A analogs modifiedat the 1-amina acid residue, wherein the stereoselective synthesis ofthe Z-isomer enriched material comprises the steps of: reacting anacetyl cyclosporin A aldehyde with [3-(diphenylphosphino)allyl] titaniumto form a titanium-containing intermediate; allowing thetitanium-containing intermediate to proceed to an erythro-α-adduct;converting the erythro-α-adduct to an β-oxidophosphonium salt bytreatment of iodomethane; converting the β-oxidophosphonium salt to anacetyl-(Z)-1,3-diene; and treating the acetyl-(Z)-1,3-diene with base toform the (Z)-isomer of ISA_(TX)247.

In still a further aspect, the invention provides mixtures (E) and(Z)-isomers prepared by a process comprising the steps of: protectingthe β-alcohol of cyclosporin A to form acetyl cyclosporin A; brominatingthe η-carbon of the side chain of the 1-amino acid residue of acetylcyclosproin A to produce a first intermediate acetyl-η-bromocyclosporinA; heating the first intermediate acetyl-η-bromocyclosporin A with areagent selected from the group consisting of triphenyl phosphine andtrialkyl phosphine to produce a second intermediate selected from thegroup consisting of triphenyl- and trialkyl phosphonium bromides ofacetyl cyclosporin A; preparing a mixture of (E) and (Z)-isomers ofacetyl-1,3-diene by stirring the ylide generated from the triphenyl- ortrialkyl salt (second intermediate triphenylphosphonium bromide) ofacetyl cyclosporin A with formaldehyde; and preparing the mixture of (E)and (Z)-isomers of ISA_(TX)247 by treating the mixture of (E) and(Z)-isomers of acetyl-1,3-diene with a base.

The invention is also directed to compositions of matter, includingtriphenyl- and trialkyl phosphonium bromides of acetyl cyclosporin A,the product prepared by a process comprising the steps of: protectingthe β-alcohol of cyclosporin A; brominating the η-carbon of the sidechain of the 1-amino acid residue of cyclosporin A to produce a firstintermediate acetyl-η-bromocyclosporin A; and heating the firstintermediate acetyl-η-bromocyclosporin A with a reagent selected fromthe group consisting of triphenylphosphine and trialkylphosphine toproduce a bromide of acetyl cyclosporin A selected from the groupconsisting of the triphenyl- and trialkylphosphonium bromides of acetylcyclosporin A. Also provided are compositions comprising a triphenyl ortrialkyl phosphonium bromide derivative of acetyl cyclosporin A andcompositions comprising a β-trimethylsilyl alcohol derivative ofcyclosporin A.

In an additional aspect, the invention provides methods for theselective preparation of cyclosporin A aldehyde comprising the steps of:protecting the β-alcohol of cyclosporin A by forming acetyl cyclosporinA or trimethylsilyl (TMS) cyclosporin A; and oxidizing the acetylcyclosporin A or TMS cyclosporin A with ozone as the oxidizing agentused with a reducing agent.

In another added aspect, the invention is directed to methods ofpreparing an isomeric mixture of cyclosporin A analogs modified at the1-amino acid residue, wherein the synthetic pathway comprises the stepsof: converting an intermediate acetyl cyclosporin A aldehyde to amixture of (E) and (Z)-isomers of acetyl-1,3-diene by reacting theintermediate with a phosphorus ylide prepared from atributylallylphosphonium halogenide via a Wittig reaction, optionally inthe presence of a lithium halide; and preparing a mixture of (E) and(Z)-isomers of ISA_(TX)247 by treating the mixture of (E) and(Z)-isomers of acetyl-1,3-diene with a base.

In an additional aspect, the invention provides methods for thestereoselective synthesis of the E-isomer of ISA_(TX)247 comprising thesteps of: reacting a trimethylsilyl (TMS) cyclosporin A aldehyde withtrialkylsilylallyl borane to form a β-trialkylsilyl alcohol; treatingthe β-trialkylsilyl alcohol to form directly the E-isomer ofISA_(TX)247.

Another aspect of the invention is directed to methods for thestereoselective synthesis of the Z-isomer of ISA_(TX)247 comprising thesteps of: reacting a trimethylsilyl (TMS) cyclosporin A aldehyde withtrialkylsilylallyl borane to form a β-trialkylsilyl alcohol; treatingthe β-trialkylsilyl alcohol with base to form TMS-(Z)-1,3-diene; anddeprotecting the TMS-(Z)-1,3-diene to form the Z-isomer of ISA_(TX)247.

The invention is also directed to methods of preparing isomeric mixturesof cyclosporin A analogs modified at the 1-amino acid residue, themethod comprising a synthetic pathway that prepares an (E)-isomer and a(Z)-isomer of ISA_(TX)247 such that the (E)-isomer and the (Z)-isomerare present in the mixture in a predetermined ratio, wherein thesynthetic pathway comprises the steps of: protecting the β alcohol ofcyclosporin A; oxidizing the protected cyclosporin A to produce a secondintermediate protected cyclosporin A aldehyde; converting the secondintermediate protected cyclosporin A aldehyde to a mixture of E- andZ-isomers of protected 1,3 diene by reacting the second intermediatewith a phosphorus ylide via a Wittig reaction, optionally in thepresence of a lithium halide; and preparing a mixture of E- andZ-isomers by deprotecting the protected 1,3 diene.

Other methods of preparing such mixtures also provided by the inventioninclude methods of preparing an isomeric mixture of cyclosporin Aanalogs modified at the 1-amino acid residue, the method comprising asynthetic pathway that prepares an (E)-isomer and a (Z)-isomer ofISA_(TX)247 such that the (E)-isomer and the (Z)-isomer are present inthe mixture in a predetermined ratio, wherein the ratio of isomers inthe mixture ranges from about 45 to 55 percent by weight of the(E)-isomer to about 55 to 45 percent by weight of the (Z)-isomer, basedon the total weight of the mixture.

The invention also provides methods of preparing a predeterminedisomeric mixture of cyclosporin A analogs modified at the 1-amino acidresidue, the method comprising: preparing a first material enriched inan (E)-isomer of ISA_(TX)247; preparing a second material enriched in a(Z)-isomer of ISA_(TX)247; and mixing the first and second materials ina ratio designed to give the desired isomeric composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the structure of cyclosporin A, illustrating the 11 aminoacid residues that comprise the cyclic peptide ring of the molecule, aswell as the structure of the side chain of the 1-amino acid residue;

FIG. 1B is another illustration of the structure of cyclosporin A withparticular emphasis on the definition of the term “CsA” as it is used inthe present description;

FIG. 2A shows the structure of the E-isomer (or trans-isomer) of thecyclosporin A analog called ISA_(TX)247;

FIG. 2B shows the structure of the Z-isomer (or cis-isomer) of thecyclosporin A analog ISA_(TX)247;

FIG. 3 shows an overview of exemplary synthetic pathways that may beused to prepare the cyclosporin analogs of the present invention, wherestereoselective pathways are grouped according to reactive conditions;

FIG. 4 illustrates a synthetic pathway that produces a mixture of (E)and (Z)-isomers of ISA_(TX)247 from a bromine precursor;

FIG. 5 illustrates another synthetic pathway that produces a mixture of(E) and (Z)-isomers of ISA_(TX)247 from an aldehyde precursor;

FIG. 6 illustrates an exemplary stereoselective reaction scheme that maybe used to prepare compositions enriched in either the (E) or(Z)-isomers of ISA_(TX)247, wherein either isomer may be prepared fromthe same precursor alcohol;

FIG. 7 illustrates an alternative reaction scheme for thestereoselective synthesis of a composition enriched in the (Z)-isomer ofISA_(TX)247;

FIG. 8 illustrates an alternative reaction scheme for thestereoselective synthesis of a composition enriched in the (E)-isomer ofISA_(TX)247;

FIGS. 9A–C illustrate exemplary synthetic pathways for producing amixture of the (E) and (Z)-isomers of ISA_(TX)247, the conditions ofeach reaction having been tailored to produce a particular exemplaryratio of the two isomers;

FIG. 10 illustrates exemplary stereoselective pathways for producing amixture of the (E) and (Z)-isomers of ISA_(TX)247, where compositionsenriched in one of the two isomers are first prepared, and then mixedaccordingly in predetermined proportions to achieve the desired ratio;

FIG. 11 provides the results of an assay which shows that the inhibitionof calcineurin phosphatase activity by ISA_(TX)247 (45–50% of E-isomerand 50–55% of Z-isomer) was up to a 3-fold more potent (as determined byIC₅₀) as compared to cyclosporine A.

FIG. 12 sets forth the structure and isomeric composition of somedeuterated and non-deuterated analogue isomeric mixtures.

FIG. 13 provides the results of an assay which shows that the inhibitionof calcineurin phosphatase activity by various deuterated andnon-deuterated analogue isomeric mixtures was at least as potent (asdetermined by IC₅₀) as compared to cyclosporine A.

DETAILED DESCRIPTION OF THE INVENTION

Synthesis

Cyclosporin and its analogs are members of a class of cyclicpolypeptides having potent immunosuppressive activity. Despite theadvantages these drugs offer with respect to their immunosuppressive,anti-inflammatory, and anti-parasitic activities, there are numerousadverse effects associated with cyclosporine A therapy that includenephrotoxicity and hepatotoxicity. Accordingly, there is a need for newimmunosuppressive agents that are as pharmacologically active as thenaturally occurring compound cyclosporin A, but without the associatedtoxic side effects.

Applicants have previously disclosed a cyclosporin A analog referred toas “ISA_(TX)247.” This analog is structurally similar to cyclosporin Aexcept for modification at the 1-amino acid residue. Applicantsdiscovered that certain mixtures of cis and trans-isomers of ISA_(TX)247exhibited a combination of enhanced potency, and reduced toxicity, overthe naturally occurring and presently known cyclosporins.

According to embodiments of the present invention, ISA_(TX)247 isomers(and derivatives thereof) are synthesized by stereoselective pathwaysthat may vary in their degree of stereoselectivity. Stereoselectivepathways produce compositions that are enriched in either of the (E) and(Z)-isomers, and these compositions may be combined such that theresulting mixture has a desired ratio of the two isomers. Alternatively,the reaction conditions of a stereoselective pathway may be tailored toproduce the desired ratio directly in a prepared mixture.

The chemical name of one immunosuppresive cyclosporin analog of thepresent invention, called ISA_(TX)247, is chemically described by thename cyclo{{E,Z)-(2S,3R,4R)-3-hydroxy-4-methyl-2-(methylamino)-6,8-nonadienoyl}-L-2-aminobutyryl-N-methyl-glycyl-N-methyl-L-Leucyl-L-valyl-N-methyl-L-leucyl-L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl}. Its empirical formula is C₆₃H₁₁₁N₁₁O₁₂, and it has a molecularweight of about 1214.85. The term “ISA_(TX)247” is a trade designationgiven to this pharmacologically active compound.

The structure of ISA_(TX)247 has been verified primarily through nuclearmagnetic resonance (NMR) spectroscopy. Both the ¹H and ¹³C spectra wereassigned using a series of one and two dimensional NMR experiments, andby comparison to the known NMR assignments for cyclosporin A. Theabsolute assignment of the (E) and (Z)-isomers of ISA_(TX)247 wasconfirmed by Nuclear Overhauser Effect (NOE) experiments. Additionalsupporting evidence was provided by mass spectral analysis, whichconfirmed the molecular weight, and by the infrared spectrum, which wasfound to be very similar to cyclosporin A. The latter result wasexpected, given the similarity between the two compounds.

The structure of cyclosporin A is illustrated in FIG. 1A. The structureincludes identification of the 11 amino acid residues that comprise thecyclic peptide ring of the molecule. These 11 amino acid residues arelabeled with numbers increasing in a clockwise direction, starting withthe amino acid shown at the top center of the ring (and identified withreference label “1-amino acid”). The first amino acid is enclosed in adashed box for clarity. The side chain of the 1-amino acid residue hasbeen drawn out chemically since it is at this general location that thesynthetic reactions described herein take place. Conventionally, thecarbon adjacent to the carbonyl group of an amino acid is labeled as theα-carbon, with progressive letters in the Greek alphabet used to labeladjacent carbons in a direction down the chain, away from the peptidering. In the case of cyclosporin A, as shown in FIG. 1A, the β-carbon ofthe side chain is bonded to a hydroxyl group, and there is atrans-oriented double bond between the ε and ζ-carbons of the sidechain.

Another schematic of the cyclosporin A structure is drawn in FIG. 1B,where a different portion of the molecule has been enclosed in a dashedbox. This figure defines the nomenclature to be used in the presentdescription, where the term “CsA” refers to the portion of thecyclosporin A enclosed in the box. The present nomenclature provides ashorthand means of displaying the region where the synthetic reactionsdescribed herein will take place (i.e., the side chain of the 1-aminoacid residue, which has been drawn outside the dashed box in FIG. 1B),without having to re-draw the remainder of the molecule each time areaction is described. It will be obvious to those skilled in the artthat the bond between the α and β-carbons of the side chain is of normallength, and has been exaggerated only in this drawing to assist with thedefinition of the term “CsA.”

As stated above, a particularly preferred cyclosporin A analog is calledISA_(TX)247, and its two stereoisomers E (or trans) and Z (or cis) areshown in FIGS. 2A and 2B, respectively. The cis or trans nature of thesestereoisomers refers to the configuration of the double bond between theε and ζ-carbons of the side chain; i.e., the double bond nearer to thepeptide ring, as opposed to the double bond at the terminal end of thechain.

A word should be said about stereochemical nomenclature. In the presentdescription the terms cis and (Z) will be used interchangeably, and theterms trans and (E) will be used interchangeably. Usage of the terms“erythro” and “threo” will be kept to a minimum due to apparentconfusion in the literature with regard to their meaning. See R. W.Hoffmann and H.-J Zei in “Stereoselective synthesis of Alcohols. 8.Diastereoselective Synthesis of β-Methylhomoallyl Alcohols viaCrotylboronates,” J. Org. Chem., Vol. 46, pp. 1309–1314 (1981); A.Streitwieser and C. H. Heathcock, Introduction to Organic Chemistry,2^(nd) ed. (Macmillan, New York, 1981), pp. 845–846; and M. B. Smith andJ. March, March's Advanced Organic Chemistry (Wiley, New York, 2001),pp. 144–147. In the few cases where threo/erythro terminology isemployed herein the convention of Streitwieser and Heathcock is used,where “erythro” isomers refer to (R,S) and (S,R) configurations, and“threo” isomers refer to (R,R) and (S,S) configurations.

A final comment about nomenclature concerns the terminal carbon-carbondouble bond shown in FIGS. 2A and 2B. In an alternate numbering scheme,the carbons in the side chain of the 1-amino acid residue may benumbered starting with the terminal (θ) carbon, and working back towardthe peptide ring. In this system the ISA_(TX)247 isomers may be thoughtof as 1,3-dienes according to conventional nomenclature in organicchemistry, where each double bond is identified by its lowest numberedcarbon.

The synthetic pathways illustrated in FIGS. 3–8 will now be discussed.According to embodiments of the present invention, isomeric mixtures maybe prepared directly, wherein the reaction conditions of a particularsynthetic pathway are tailored to achieve the desired ratio of isomersin the mixture. Alternatively, compositions may be prepared that areenriched in one of the two geometrical isomers of a cyclosporin Aanalog, and the compositions combined in a predefined ratio to achievethe desired mixture.

An overview of the synthetic pathways according to embodiments of thepresent invention is given in FIG. 3, where particular emphasis is givento grouping reaction paths according to chemistry and stereoselectivity.Referring to FIG. 3, synthetic pathways that utilize Wittig reactionsare shown generally on the right-hand side of the diagram as indicatedby reference numeral 31, while pathways 32 and 33 that utilizeorganometallic reagents that are thought to form six-membered ringtransition states are shown in the middle and left-hand sides of thediagram. Any of the synthetic pathways may yield a mixture of theisomers, or they may produce compositions enriched in one of the twoisomers.

Embodiments of the present invention provide a variety of ways to arriveat the desired mixture of isomers. The flexibility and versatility ofthe synthetic strategies disclosed herein may be reflected in part bythe symmetries and asymmetries of FIG. 3. A reaction that is common toeach of the pathways is the protection of a functional group incyclosporin A 34; in this exemplary embodiment that reaction is theconversion of cyclosporin A 34 to acetyl cyclosporin A 35. An asymmetryin FIG. 3 is the use of acetyl cyclosporin A aldehyde compound 51 as aprecursor for all of the titanium and lithium organometallic reagentpathways, but only some of the phosphorus containing Wittig reactionpathways.

In general, synthetic pathways of FIG. 3 whose reaction conditions maybe tuned to produce a mixture having the desired ratio of isomersutilize phosphorus-containing reagents as participants in a Wittigreaction. Other stereoselective pathways make use of inorganic elementsas well, typically as members of organometallic reagents that proceedthrough six-membered ring transition states where steric hindrancedictates the configurational outcome. A plethora of organometallicreagents are useful to the present invention, including those thatfeature inorganic elements such as boron, silicon, titanium, lithium,and sulfur.

Compositions enriched in one or the other of a pair of isomers may beprepared from a single precursor; alternatively, the two compositionsmay be prepared from different precursors. In one of the stereoselectivepathways of FIG. 3 (pathway 32), a single precursor leads to both of thetwo isomers of ISA_(TX)247, depending on the reaction conditions thatare chosen. In another of the stereoselective pathways (pathway 33), twodifferent precursors are needed to produce each of the enrichedcompositions.

The reactions of FIG. 3 will now be discussed in detail. A reaction thatis common to each of the pathways is the protection of the alcohol atthe β-position of the side chain of the 1-amino acid residue. Such aprotection scheme addresses a problem commonly encountered in organicsynthesis, where a first functional group is inadvertently modified by areaction intended for a second (similar and/or identical) functionalgroup located elsewhere on the molecule. To carry out the scheme thefirst functional group is reacted with a protecting group, the desiredreaction is carried out on the second functional group, and theprotecting group is then removed from the first functional group.

Protecting groups are well known in organic synthesis, and have beendiscussed by J. R. Hanson in Chapter 2, “The Protection of Alcohols,” ofthe publication Protecting Groups in Organic Synthesis (SheffieldAcademic Press, Sheffield, England, 1999), pp. 24–25. Hanson teaches howto protect hydroxyl groups by converting them to either esters orethers. Acetate esters are perhaps the most frequently used type ofchemistry for protecting hydroxyl groups. There are a wide range ofconditions that may be used to introduce the acetate group. Thesereagents and solvents include acetic anhydride and pyridine; aceticanhydride, pyridine and dimethylaminopyridine (DMAP); acetic anhydrideand sodium acetate; acetic anhydride and toluene-p-sulfonic acid, acetylchloride, pyridine and DMAP; and ketene. DMAP is a useful acylationcatalyst because of the formation of a highly reactive N-acylpyridiniumsalt from the anhydride.

In one embodiment of the present invention, the β-alcohol of cyclosporinA 34 is protected as an acetate by reacting 34 with acetyl chloride,ethyl acetate, or combinations thereof, forming the compound acetylcyclosporin A 35. In another embodiment, the β-alcohol undergoes anucleophilic addition to acetic anhydride, forming acetyl cyclosporin A35 and acetic acid. These reactions may be carried out in the presenceof dimethylaminopyridine (DMAP) where an excess of acetic anhydride actsas the solvent. In these cases the prefix “acetyl” may be used in thenomenclature throughout the synthetic pathway, or until the acetyl groupis removed. For example, the last intermediate in one pathway having anacetyl group at the β-carbon is called “acetyl-(E)-1,3-diene.”

Although the preparation of acetyl cyclosporin A is well established inthe literature, it will be appreciated by those skilled in the art thatprotecting groups other than acetate esters may be used to protect theβ-alcohol of the 1-amino acid residue of cyclosporin A 34. Theseprotecting groups may include benzoate esters, substituted benzoateesters, ethers, and silyl ethers. Under certain reaction conditions, theacetate protecting group is prone to undesirable side reactions such aselimination and hydrolysis. Since benzoate esters, ethers and silylethers are often more resistant to such side reactions under those samereaction conditions, it is often advantageous to employ such protectinggroups in place of acetate. Cyclosporin or cyclosporin derivatives whichhave been protected by an acetyl group or any other protecting group arereferred to as “protected-cyclosporin A.” Likewise, the ultimateintermediate in the exemplary pathway referred to above would be called“protected-(E)-1,3-diene” instead of “acetyl-(E)-1,3-diene.” The natureof the chosen protecting group may have an influence on the desiredcourse of further steps in the reaction sequences.

Referring to FIG. 3, acetyl cyclosporin A 35 has in this exemplarypathway a protected β-alcohol, and this compound serves as a precursorfor the synthesis of ISA_(TX)247 isomers in several of the syntheticpathways. Wittig reaction pathways will be discussed first.

Synthesis of Mixtures of the (E) and (Z)-Isomers of ISA_(TX)247 Via theWittig Reaction

Wittig reaction pathways exemplified herein are identified by thereference numeral 31 in FIG. 3. Method 1 proceeds through the bromineintermediate acetyl-η-bromocyclosporin 41, whereas method 2 utilizes theacetyl cyclosporin A aldehyde 51 as a starting point. The exemplarymethods described below utilize a Wittig reaction to introduce an alkenefunctionality with a mixture of stereochemical configurations.

The Wittig reactions used in the exemplary embodiments disclosed hereinto synthesize mixtures of the (E) and (Z)-isomers of ISA_(TX)247 mayoptionally be carried out in the presence of a lithium halide. Thepresence of lithium halides in Wittig reactions is well known to have aneffect on the ratio of geometrical isomers produced and, therefore, theaddition of such a compound can aid in producing a desired mixture ofthe (E) and (Z)-isomers of ISA_(TX)247.

Method 1

In one embodiment of the present invention, a mixture of (E) and(Z)-isomers of ISA_(TX)247 is prepared as shown in FIG. 4. The use ofthe wavy-lined representation in FIG. 4 (see especially compounds 43 and44) is meant to denote that the exemplary reaction sequence produces amixture of (E) and (Z)-isomers. In one embodiment the percentage ratioof the (E) to (Z)-isomers produced ranges from about 10 to 90 percent ofthe (E)-isomer to about 90 to 10 percent of the (Z)-isomer, but theseranges are only exemplary, and many other ranges are possible. Forexample, the mixture may contain from about 15 to 85 percent by weightof the (E)-isomer and about 85 to 15 percent of the (Z)-isomer. In otherembodiments, the mixture contains about 25 to 75 percent by weight ofthe (E)-isomer and about 75 to 25 percent by weight of the (Z)-isomer;about 35 to 65 percent by weight of the (E)-isomer and about 65 to 35percent by weight of the (Z)-isomer; and about 45 to 55 percent byweight of the (E)-isomer and about 55 to 45 percent of the (Z)-isomer.In still another embodiment, the isomeric mixture is an ISA_(TX)247mixture which comprises about 45 to 50 percent by weight of the(E)-isomer and about 50 to 55 percent by weight of the (Z)-isomer. Thesepercentages by weight are based on the total weight of the composition,and it will be understood that the sum of the weight percent of the (E)isomer and the (Z) isomer is 100 weight percent. In other words, amixture might contain 65 percent by weight of the (E)-isomer and 35percent by weight of the (Z)-isomer, or vice versa.

Referring to FIG. 4, the terminal η-carbon of the side chain of the1-amino acid residue of acetyl-cyclosporin A is brominated in the nextstep of the reaction by refluxing acetyl cyclosporin A 35 withN-bromosuccinimide and azo-bis-isobutyronitrile in a solvent such ascarbon tetrachloride, producing the intermediateacetyl-η-bromocyclosporin A 41. N-bromosuccinimide is a reagent that isoften used to replace allylic hydrogens with bromine, and it is believedto do so via a free radical mechanism. The preparation of theintermediate 41 was essentially described by M. K. Eberle and F.Nuninger in “Synthesis of the Main Metabolite (OL-17) of Cyclosporin A,”J. Org. Chem., Vol. 57, pp. 2689–2691 (1992).

The novel intermediate triphenylphosphonium bromide of acetylcyclosporin A 42 may be prepared from acetyl-η-bromocyclosporin A 41 byheating the latter compound with triphenylphosphine in a solvent such astoluene.

The novel intermediate 42, and others like it, are contemplated to bekey intermediates in the synthesis of a plurality of cyclosporin Aanalogs that contain a conjugated diene system in the 1-amino acidresidue. For example, in addition to triphenylphosphine, compounds suchas triarylphosphines, trialkylphosphines, arylalkylphosphines, andtriarylarsines may be reacted with acetyl-η-bromocyclosporin A 41 toprepare other activated compounds similar to 42.

Referring again to FIG. 4, a mixture of the (E) and (Z)-isomers ofacetyl-1,3-diene 43 may be prepared by stirring the triphenylphosphoniumbromide of acetyl cyclosporin A 42 with an excess of formaldehyde intoluene at room temperature. Following addition of the formaldehyde, abase such as sodium hydroxide is added dropwise, and the isomericmixture of dienes is extracted with ethyl acetate.

Numerous organic chemistry textbooks describe the Wittig reaction. Onedescription in particular is provided by J. McMurry, Organic Chemistry,5^(th) Ed. (Brooks/Cole, Pacific Grove, 2000), pp. 780–783. A Wittigreaction may be used to convert a ketone or an aldehyde to an alkene. Insuch a process, a phosphorus ylide, also called a phosphorane, may bereacted with the aldehyde or ketone to give a dipolar intermediatecalled a betaine. Typically the betaine intermediate is not isolated;rather, it spontaneously decomposes through a four-membered ring toyield an alkene and triphenylphosphine oxide. The net result is areplacement of the carbonyl oxygen atom by the R₂C═ group originallybonded to the phosphorus.

It will be appreciated by those skilled in the art that a wide varietyof reagents may be substituted for the exemplary Wittig reactionreagents cited above. For example, numerous alkyl, aryl, aldehyde, andketone compounds may be substituted for formaldehyde to prepare a vastnumber of cyclosporin derivatives. Applicants have carried out the abovesynthesis with formaldehyde, and in place of formaldehyde, compoundssuch as acetaldehyde, deuterated formaldehyde, deuterated acetaldehyde,2-chlorobenzaldehyde, benzaldehyde, and butyraldehyde. Such Wittigreactions may be carried out with compounds other thantriphenylphosphonium derivatives, such as triarylphosphines,trialkylphosphines, arylalkylphosphines and triarylarsines. Instead ofusing sodium hydroxide, various other bases such as sodium carbonate,butyllithium, hexyllithium, sodium amide, lithium hindered bases such aslithium diisopropylamide, and alkali metal alkoxides may be used. Inaddition to varying these reagents, the reaction may be conducted invarious organic solvents or mixtures of organic solvents and water, inthe presence of various salts, particularly lithium halides, and atvarying temperatures. All of the factors listed above can reasonably beselected by one of ordinary skill in the art to have the desired effecton the stereochemistry of the formed double bond; i.e., the desiredeffect on the ratio of the cis to trans-isomers. In one embodiment ofthe present invention, the Wittig reaction is carried out in a solventselected from the group consisting of tetrahydrofuran and toluene, andwherein the solvent is used in the presence of a compound selected fromthe group comprised of butyllithium, sodium lower alkoxide, potassiumlower alkoxide, and carbonate at a temperature between about −80° C. and110° C. The potassium lower oxide may be a potassium-tert-butoxide.Futhermore, the solvent may be tetrahydrofuran used in the pressence ofpotassium-tert-butoxide at a temperature between about −70° C. and −100°C.

In a final step of this synthesis, the protecting group on the β-carbonis removed using the following procedure. The mixture ofacetyl-(E)-1,3-diene and acetyl-(Z)-1,3-diene 43 is dissolved inmethanol, and then water is added. A base such as potassium carbonate isadded, and the reaction mixture stirred at room temperature. Bases otherthan potassium carbonate that may be used include sodium hydroxide,sodium carbonate, sodium alkoxide, potassium alkoxide and amine basesselected from the group consisting of NH₃, hydroxylamine, hydrazine, andlower dialkylamines. Ethyl acetate is then used to extract the finalproduct mixture of (E) and (Z)-isomers of ISA_(TX)247 44.

Method 2

In an alternative reaction pathway for synthesizing a mixture of (E) and(Z)-isomers of ISA_(TX)247 via a Wittig reaction strategy, a four stepsynthetic pathway may be employed as follows: 1) protection of theβ-alcohol, as in method 1, 2) oxidation of the acetyl-cyclosporin Aproduced from the first step to produce an aldehyde; 3) a Wittigreaction; and 4) de-acetylation of the Wittig reaction product, orequivalently, hydrolysis of the acetate ester to retrieve the alcohol.This reaction sequence is illustrated in FIG. 5.

This synthetic pathway begins in a manner similar to the Wittig reactionpathway of FIG. 4 in that the first step protects the β-alcohol with anacetate ester group. The two pathways differ from here on, however, inthat the next step of method 2 converts acetyl-cyclosporin A 35 to analdehyde, acetyl cyclosporin A aldehyde 51. This reaction uses anoxidizing agent sufficiently strong to cleave a C═C bond to produce twofragments. Alkene cleavage is known in the art. Ozone is perhaps themost commonly used double bond cleavage reagent, but other oxidizingreagents such as potassium permanganate (KMnO₄) or osmium tetroxide cancause double bond cleavage as well.

According to an embodiment of the present invention, acetyl-cyclosporinA is converted to an aldehyde with ozone as the oxidizing agent followedby work-up with a reducing agent to form acetyl cyclosporin A aldehyde.The ozonolysis step is carried out at a temperature range from about−80° C. to 0° C. The solvent used during the ozonolysis may be a loweralcohol such as methanol. The reducing agent may be a triakylphosphinesuch as tributyl phosphine, a triarylphosphine, a trialkylamine such astriethylamine, an alkylaryl sulfide, a thiosulfate or a dialkylsulfidesuch as dimethylsulfide. When working with tributylphosphine as thereducing agent, the person of ordinary skill in the art will know thatthe reaction is dose-controlled.

According to another embodiment of the present invention, the β alcoholof cyclosporin A is protected with a trimethylsilyl (TMS) group andoxidized with ozone as the oxidizing agent followed by work-up with areducing agent to form TMS cyclosporin A aldehyde. The ozonolysis stepis carried out at a temperature range from about −80° C. to 0° C. Thesolvent used during the ozonolysis may be a mixture of lower alcohol anddichloromethane. The reducing agent may be selected from the groupconsisting of triakylphosphines such as tributyl phosphine,triarylphosphines, trialkylamines such as triethylamine, alkylarylsulfides, thiosulfates or dialkylsulfides such as dimethylsulfide. Whenworking with tributylphosphine as the reducing agent, the person ofordinary skill in the art will know that the reaction isdose-controlled.

Additionally, the cyclosporin A aldehyde can be prepared by protectingthe β-alcohol of cyclosporin A by forming acetyl cyclosporin A and thenconverting the acetyl cyclosporin A to the acetyl cyclosporin A epoxidewith a monopersulfate, preferably OXONE™ (Du Pont), in the presence of aketone, preferably an activated ketone such as acetoxyacetone ordiacetoxyacetone. This step is performed in an organic solvent which isinert under these reaction conditions such as acetonitrile and water.Ethylenediamintetra-acetic acid disodium salt is added to capture anyheavy metal ions which might be present. The epoxidation reaction iscarried out preferably at a pH over 7. This epoxidation reaction isfollowed by oxidative cleavage of the epoxide with periodic acid orperodate salt under acidic conditions. Optionally, the oxidation and theoxidative cleavage can be combined in a work-up procedure. Thesereactions have been discussed by Dan Yang, et al., in “A C₂ SymmetricChiral Ketone for Catalytic Asymmetric Epoxidation of UnfunctionalizedOlefins,” J. Am. Chem. Soc., Vol. 118, pp. 491–492 (1996), and “NovelCyclic Ketones for Catalytic Oxidation Reactions,” J. Org. Chem., Vol.63, pp. 9888–9894 (1998).

The use of ruthenium based oxidizing agents has been discussed by H. J.Carlsen et al. in “A Greatly Improved Procedure for Ruthenium TetroxideCatalyzed Oxidations of Organic Compounds,” J. Org. Chem., Vol. 46, No.19, pp 3736–3738 (1981). Carlsen et al. teach that, historically, theexpense of ruthenium metal provided an incentive for the development ofcatalytic procedures, the most popular of which used periodate orhypochlorite as stoichiometric oxidants. These investigators found aloss of catalytic activity during the course of the reaction with theconventional use of ruthenium which they postulated to be due to thepresence of carboxylic acids. The addition of nitriles to the reactionmixture, especially acetonitrile, was found to significantly enhance therate and extent of the oxidative cleavage of alkenes in a CCl₄/H₂O/IO₄ ⁻system.

According to one embodiment of the present invention, acetyl cyclosporinA aldehyde 51 may be produced from acetyl cyclosporin A 35 by dissolvingit in a mixture of acetonitrile and water, and then adding first sodiumperiodate and then ruthenium chloride hydrate. The aldehyde 51 may beextracted with ethyl acetate. It should be noted that the synthesis ofthe aldehyde 51 by this oxidative cleavage strategy is important to manyof the stereoselective pathways to be discussed below, and consequentlythe reader is referred back to this section accordingly.

The third step of method 2 involves converting the aldehyde 51 to amixture of (E) and (Z) dienes via a Wittig reaction, in a similarfashion to that of method 1. As in method 1, a phosphorus ylide adds tothe aldehyde to yield a betaine (which is not isolated), with the netresult that the carbonyl oxygen atom of the aldehyde is replaced by theR₂C═ group originally bonded to phosphorus. Again, such Wittig reactionsmay be carried out with phosphorus containing compounds other thantriphenylphosphonium derivatives, such as triarylphosphines,trialkylphosphines, arylalkylphosphines and triarylarsines, at varioustemperatures, and using a variety of basic solutions and solvents or theaddition of various inorganic salts may be used to influence thestereochemistry of the newly formed double bond.

In one embodiment, acetyl cyclosporin A aldehyde 51 is dissolved intoluene, to which is added a base such as sodium hydroxide in water.Allyl triphenylphosphonium bromide 52 is then added, and the reactionstirred for some time. Workup of the product mixture of acetyl (E) and(Z)-1,3-dienes 53 involves extraction with hexane and/or ethyl acetate,where the term “workup” is intended to mean the process of extractingand/or isolating reaction products from a mixture of reactants,products, solvent, etc.

In a final step of method 2, similar to the final step of method 1, theacetate ester group protecting the alcohol at the β-carbon position isremoved with potassium carbonate, yielding a mixture of (E) and (Z)isomers of ISA_(TX)247 54. Bases other than potassium carbonate that maybe used to remove the protecting group include sodium hydroxide, sodiumcarbonate, sodium alkoxide, potassium alkoxide and amine bases selectedfrom the group consisting of NH₃, hydroxylamine, hydrazine, and lowerdialkylamines.

Synthesis of Compositions Enriched in Either of the ISA_(TX)247 (E) and(Z)-isomers Via Organometallic Routes

According to embodiments of the present invention, stereoselectivesynthetic pathways may employ the use of inorganic reagents containingelements such as silicon, boron, titanium, sulfur, phosphorus, and/orlithium. These pathways may proceed through a six-membered ringtransition state where one of the members of the ring is the inorganicelement from the organometallic reagent. In some embodiments, sterichindrance effects related to the transition state may influence thestereochemical outcome of the reaction.

Two exemplary stereoselective schemes will be discussed in the presentdisclosure. In the first stereoselective scheme (method 3, also shown asPathway 32 in FIG. 3), a silicon-containing compound undergoes anelimination reaction to produce either the (E) or (Z)-isomer, dependingon whether the elimination reaction is carried out under acidic or basicconditions. This is an example of a Peterson olefination. In the secondstereoselective scheme (method 4, also shown as Pathway 33 in FIG. 3),each of the isomers is produced from a different precursor. The(Z)-isomer is produced from a titanium and phosphorus containingintermediates, whereas the (E)-isomer is produced through a lithiumcontaining intermediate.

Method 3

This pathway proceeds via the acetyl cyclosporin A aldehyde 51.

A similar reaction scheme has been discussed in general by D. J. S. Tsaiand D. S. Matteson in “A Stereocontrolled Synthesis of (Z) and (E)Terminal Dienes from Pinacol (E)-1-Trimethylsilyl-1-Propene-3-Boronate,”Tetrahedron Letters, Vol. 22, No. 29, pp. 2751–2752 (1981). The methodis illustrated in FIG. 6. In general, the synthesis involves preparing atrimethylsilylallylboronate ester reagent 62, and then treating acetylcyclosporin A aldehyde 51 with 62 to form a β-trimethylsilyl alcohol 64.This alcohol is believed to form via a boron-containing transition state63. As the boronate esters are slow-reacting in allylboration reactions,it will be appreciated by those skilled in the art that the use of afaster-reacting borane reagent such as E-γ-trimethylsilyl diethylboraneor 9-(E-γ-trimethylsilylallyl)-9-BBN has advantages. Theβ-trimethylsilyl alcohol 64 may then undergo a Peterson olefination toprepare an alkene, in this case either the diene 65 or the diene 67.

Formation of the alkene follows one of two distinct paths, depending onwhether the elimination reaction (the olefination) is carried out underacidic or basic conditions. Under acidic conditions an anti-eliminationoccurs forming the (E)-isomer, whereas under basic conditions acis-elimination occurs to form the (Z)-isomer. It will be appreciated bythose skilled in the art that by using this synthetic pathway, eitherisomer may be prepared from the same precursor. The product of eachelimination reaction comprises a composition enriched in one of the twoisomers. In one embodiment, enriched means that the composition containsgreater than or equal to about 75 percent by weight of an isomer. Inother embodiments, the enriched composition my comprise 80, 85, and 90percent by weight of one of the isomers. The compositions enriched in anisomer may then be combined in a predetermined ratio to arrive at thedesired mixture as illustrated in FIG. 10.

The reactions in FIG. 6 will now be discussed in detail, beginning withthe preparation of the boron-containing reagent 62. A generalinvestigation of the use of silicon reagents in the synthesis ofcarbon-carbon bond forming reactions has been discussed by E. Ehlingerand P. Magnus in “Silicon in Synthesis. 10. The (Trimethylsilyl)allylAnion: β-Acyl Anion Equivalent for the Conversion of Aldehydes andKetones into γ-Lactones,” J. Am. Chem. Soc., Vol. 102, No. 15, pp.5004–5011 (1980). In particular, these investigators teach the reactionbetween the (trimethylsilyl)allyl anion and an aldehyde. The anion maybe prepared by deprotonating allyltrimethylsilane with sec-butyllithiumin tetrahydrofuran at −76° C. containing 1 equivalent oftetramethylethylenediamine (TMEDA).

The deprotonation of allyltrimethylsilane (this step is not shown inFIG. 6) has been discussed by J.-F. Biellmann and J.-B. Ducep in“Allylic and Benzylic Carbanions Substituted by Heteroatoms,” OrganicReactions, Vol. 27 (Wiley, New York, 1982), p. 9. A proton alpha to theheteroatom in substituted allylic systems may be removed with a morebasic agent. A large variety of such agents are available, with perhapsn-butyllithium being the most common. n-Butyllithium is used in astoichiometric amount with the compound to be metalated in solution withtetrahydrofuran (THF). The temperature is usually maintained below 0° C.(often below −76° C.) where the n-butyllithium has a low reactivity dueto its polymeric nature. Addition of a chelating agent such asN,N,N′,N′-tetramethylethylenediamine (TMEDA) causes the polymer todissociate. However, the reaction can also be done at room pemperature,even in the absence of TMEDA.

Allylsilanes are easily deprotonated because the anion that is generatedis stabilized not only through conjugation with the adjacent doublebond, but also by the neighboring silyl group. The anion may react withelectrophiles through either its α-carbon or its γ-carbon. Theregiochemical and stereochemical outcome of these reactions depends onseveral factors, one of the most important of which is the identity ofthe counterion. See the discussion of allylsilanes by S. E. Thomas inOrganic Synthesis: The Roles of Boron and Silicon (Oxford UniversityPress, New York, 1991), pp. 84–87.

Thus the products of the stereospecific elimination of a β-hydroxyalkylsilane are the acetyl-(E)-1,3-diene compound 67 and theacetyl-(Z)-1,3-diene compound 65. As in the previous methods, theprotecting group may now be removed from each of these dienes bytreatment with K₂CO₃ in methanol and water. This removes the acetategroup bonded to the β-carbon of the 1-amino acid residue, returning thefunctional group on that carbon to an alcohol. Bases other thanpotassium carbonate that may be used to remove the protecting groupinclude sodium hydroxide, sodium carbonate, sodium alkoxide, potassiumalkoxide and amine bases selected from the group consisting of NH₃,hydroxylamine, hydrazine, and lower dialkylamines.

The addition of an aldehyde to an allylborane has been discussed by S.E. Thomas in the above reference at pages 34–35. The addition of analdehyde to an allylborane, wherein the latter is unsymmetricallysubstituted at the distal end of the carbon-carbon double bond (“distal”meaning furthest away from the boron atom) produces a homoallylicalcohol containing two adjacent chiral centers. (E)-allylboranes giverise to the threo-diastereoisomer, while (Z)-allylboranes give rise tothe erythro-diastereoisomer. An exemplary reaction of an (E)-allylborane62 with cyclosporin A aldehyde 51 is shown in FIG. 6, where the boronintermediate 63 is formed after stirring the reactants in a THF solutionfor a period of several days.

The reference numeral 69 in the boron intermediate 63 (FIG. 6) is meantto indicate that any number of structures are possible at the boronposition. For example, if the boronate reagent 62 is atrialkylsilylallyl boronate ester, then the structure at 69 wouldcomprise a 5-membered ring that includes two oxygen atoms. Substitutionson the boronate or borane reagents employed in 62 will be present in thestructure in 63.

It has been postulated that the stereoselectivity that is achieved inreactions involving allylboranes with aldehydes may be due to thesix-membered ring chair-like transition state exemplified by the boronintermediate 63, and depicted in FIG. 6. Only the two carbonyl atoms ofthe aldehyde (the carbon and the oxygen which are double bonded) becomemembers of the six-membered ring transition; the remainder of thealdehyde extends off the ring. The CsA portion of the aldehyde thatextends away from the six-membered ring is postulated to exist in anequatorial rather than axial position relative to the ring because thelatter configuration would give rise to unfavorable steric hindrancebetween that substituent and an oxygen atom of the allylborane 62. Itwill also be appreciated by those skilled in the art that the positionof the SiMe₃ group from the (trimethylsilyl)allyl anion is shownoccupying an equatorial position in FIG. 6 because this example startedwith the (E)-diastereomer of the allylborane. Alternatively, the SiMe₃group could have been drawn in an axial position if the startingallylborane had been the (Z)-diastereomer.

Alternatively, it is contemplated to prepare the erythro-silyl alcohol,for which acid elimination would give the cis-isomer and baseelimination would give the trans-isomer, in an opposite manner to theelimination reactions discussed above. It will be obvious to thoseskilled in the art that the same products would be obtained at the endof the synthesis.

Treatment of the transition state product 63 with triethanolamine yieldsthe β-trimethylsilyl alcohol 64. On the other hand, allylborationproduct of (trimethylsilylallyl)dialkyl borane yields silyl alcohol 64upon oxidation using NaOH/H₂O₂ or aqueous workup. The alcohol 64depicted in FIG. 6 is the threo-diastereoisomer, since the transitionstate allylborane 63 was in the (E)-configuration, although it will beappreciated by those skilled in the art that the other diastereoisomerecould have been prepared as well if starting from the Z-allylboranereagent. The diastereoselectivity in the newly created chiral centers isnot determined at this stage due to removal of these chiral centers at alater stage of the synthesis. The structure of the β-trimethylsilylalcohol 64 shown in FIG. 6 has been confirmed by the applicants usingspectral techniques.

In a method of alkene synthesis known as a Peterson olefination,elimination of the trialkylsilyl group and the hydroxy group from theβ-trimethylsilyl alcohol 64 leads to an alkene; in this case a diene,due to the double bond that is already present between the two terminalcarbons of the chain. A discussion of the conversion of β-hydroxysilanesto alkenes has been presented in the S. E. Thomas reference at pages68–69. A further discussion of this reaction is presented by P. F.Hurdlik and D. Peterson in “Stereospecific Olefin-Forming EliminationReactions of β-Hydroxysilanes,” J. Am. Chem. Soc., Vol. 97, No. 6, pp.1464–1468 (1975).

Referring to FIG. 6, the elimination reaction converting the alcohol 64to a diene may follow one of two distinct mechanistic pathways dependingon whether the reaction is carried out under acidic or basic conditions.One pathway leads to the diene 65, while the other pathway leads to thediene 67. Under acidic conditions anti-elimination occurs, while underbasic conditions syn-elimination occurs. In other words, the eliminationreactions of β-hydroxysilanes are stereospecific, and the acid- andbase-promoted reactions take the opposite stereochemical course. Typicalacids for the acid-promoted reaction may include acetic acid, sulfuricacid and various Lewis acids; typical bases include sodium hydride andpotassium hydride or potassium tert-butoxide. It may be the case thatelimination reactions using sodium hydride in THF are slow at roomtemperature, while elimination reactions that use potassium hydride takeplace more readily.

The stereospecificity occurs at this stage of the reaction pathwaybecause elimination under acidic conditions requires the trimethylsilyland hydroxy groups to be in an antiperiplanar relationship. In contrast,elimination under basic conditions requires that the trimethylsilyl andhydroxy groups adopt a synperiplanar relationship. The latter conditionfacilitates the formation of a strong silicon-oxygen bond and anintermediate four-membered ring, which breaks down in a manner analogousto the final step of a Wittig reaction. It will be appreciated by thoseskilled in the art that a strong silicon-oxygen bond replaces a weakersilicon-carbon bond, which overrides the replacement of a strongcarbon-oxygen bond with a weaker carbon-carbon π bond.

Thus the products of the stereospecific elimination of a β-hydroxyalkylsilane are the acetyl-(E)-1,3-diene compound 67 and theacetyl-(Z)-1,3-diene compound 65. As in the previous methods, theprotecting group may now be removed from each of these dienes bytreatment with K₂CO₃ in methanol and water. This removes the acetategroup bonded to the β-carbon of the 1-amino acid residue, returning thefunctional group on that carbon to an alcohol. Bases other thanpotassium carbonate that may be used to remove the protecting groupinclude sodium hydroxide, sodium carbonate, sodium alkoxide, andpotassium alkoxide.

At this stage of the preparation the synthesis is substantiallycomplete. The compositions enriched in one or the other of the isomersmay be mixed to achieve the desired ratio of isomers in the mixture. By“enriched” is meant a product that comprises at least about 75 percentby weight of that isomer; in other words, the product may contain up to25 percent by weight of the “undesired” isomer. The mixture is designedto achieve the desired pharmacological result.

Method 4

This pathway also proceeds via the acetyl cyclosporin A aldehyde 51.

An alternate scheme for producing stereoselective isomers is illustratedin FIGS. 7–8. This synthetic pathway differs from those previouslydiscussed, in that 1) the synthetic pathway for producing the (E)-isomerof ISA_(TX)247 proceeds through different intermediates than that forthe (Z)-isomer, and 2) these synthetic pathways make use of titanium andlithium-containing reagents and/or intermediates.

Titanium reagents are known to be particularly useful in organicsynthesis because they are regio- and stereoselective in their reactionswith aldehydes and ketones. The general nature of titanium instereoselective chemistry has been discussed by M. T. Reetz inOrganotitanium Reagents in Organic Synthesis (Springer-Verlag, Berlin,1986), pp. VII, 148–149, and 164–165. Here it is stated that the natureof the titanium ligand may be varied such that the electronic and stericidentity of the reagent can be manipulated, and the stereochemicaloutcome of many C—C bond forming reactions may be predicted. Accordingto this chemistry, the union of two prochiral centers of achiralmolecules creates two centers of chirality. A general rule governing thestereoselective outcome is that Z-configured enolates or crotyl metalcompounds preferentially form syn-adducts, while E-configured reagentsfavor the anti-diastereomers. The trends may again be explained byassuming a six-membered cyclic transition state having a chair geometry.

A specific example of this type of stereoselective synthesis has beendiscussed by Y. Ikeda et al. in “Stereoselective Synthesis of (Z)- and(E)-1,3-Alkadienes from Aldehydes Using Organotitanium and LithiumReagents,” Tetrahedron, Vol. 43, No. 4, pp. 723–730 (1987). Thisreference discloses that allyldiphenylphosphine may be used to produce a[3-(Diphenylphosphino)allyl]titanium reagent, which in turn may becondensed with an aldehyde followed by phosphonium salt formation togive a (Z)-1,3-alkadiene in a highly regio- and stereoselective manner.In contrast, a lithiated allyldiphenylphosphine oxide can condense withan aldehyde to give an (E)-1,3-alkadiene directly, again with thedesired stereoselectivity.

Referring to FIG. 7, synthesis of the (Z)-isomer of ISA_(TX)247 proceeds(as in the previous schemes) by generating acetyl cyclosporin A aldehyde51 from cyclosporin A 34. The [3-(diphenylphosphino)allyl]titaniumreagent 72 is prepared by deprotonating allyldiphenylphosphine 71 with astrong base such as t-BuLi, and then reacting the product with titaniumtetraisopropoxide. A transition state 73 is theoretically proposedleading to the erythro-α-adduct 74, which then may be converted to theβ-oxidophosphonium salt 75 by treatment of 74 with iodomethane (MeI). Itis postulated that the existence of the transition state 73 is at leastin part responsible for the stereoselectivity of this synthetic pathway.

In accordance with the exemplary methods outlined in the presentdisclosure, the metal site of the organometallic reagent may be theentity that controls regioselectivity (Ikeda, p. 725). This means thatthe aldehyde 51 in FIG. 7 reacts with the diphenylphosphino compound 72at its α-position to give the corresponding α-adduct 74, since theγ-carbon of the diphenylphosphino group is coordinated to the metal,which in this case is titanium. The observed Z selectivity of the dieneproduct is explained by considering the six-membered transition state73. Since both the bulky cyclosporin A side chain of the aldehyde 35 andthe diphenylphosphino group are postulated to occupy equatorialpositions in the transition state, the erythro α-adduct 74 isselectively formed, giving rise to the (Z)-1,3-diene 76.

In contrast to the reaction pathway depicted in FIG. 7, in which the(Z)-isomer of ISA_(TX)247 is produced via a titanium transition state,the (E)-isomer is not as easily produced by this method. In fact,attempts to synthesize the (E)-isomer by this method are generallyreported to result in low yields. Instead, as shown in FIG. 8, thelithio derivative 82 may be reacted with the aldehyde 51 to produce thelithium containing transition state 83, which forms the 1,3-diene in E/Zratios in a range greater than approximately 75:25. As in FIG. 7, thehigh stereoselectivity of the reaction product is possibly due to thetransition state 83, in which the vinyl group of the lithium reagent 82and the cyclosporin A side chain of the aldehyde 51 are postulated tooccupy equatorial positions, thereby producing the (E)-1,3-diene 84 in astereoselective manner. As discussed previously, certain undesirableside-reactions involving the acetate protecting group may be avoided inall stereoselective syntheses through the use of protecting groups suchas benzoate esters or silyl ethers.

Preparation of Mixtures

As stated previously, certain mixtures of cis and trans-isomers ofISA_(TX)247 were found to exhibit a combination of enhanced potencyand/or reduced toxicity over the naturally occurring and presently knowncyclosporins.

According to embodiments of the present invention, ISA_(TX)247 isomers(and derivatives thereof) are synthesized by stereoselective pathwaysthat may vary in their degree of stereoselectivity. Stereoselectivepathways may produce a first material or composition enriched in the(E)-isomer, and a second material or composition enriched in the(Z)-isomer, and these materials may then be combined such that theresulting mixture has a desired ratio of the two isomers. Alternatively,it is contemplated that the first material may be prepared by separatinga reaction product to isolate and enrich the (E)-isomer, and the secondmaterial may be prepared by separating a reaction product to isolate andenrich the (Z)-isomer. In yet another embodiment, the reactionsconditions of a stereoselective pathway may be tailored to produce thedesired ratio directly in a prepared mixture.

These principles are illustrated in FIGS. 9A–C and 10. In FIGS. 9A–C,three hypothetical synthetic reactions are shown that produce ratios ofthe (E) to the (Z)-isomer of approximately 65 to 35 percent by weight,50 to 50 percent by weight, and 35 to 65 percent by weight,respectively. Of course, these ratios are exemplary and for illustrativepurposes only, and any hypothetical set of numbers could have beenchosen. It will be obvious to those skilled in the art that the reactionconditions used to produce the ratio in FIG. 9A may be different fromthose of FIGS. 9B and 9C in order to achieve a different ratio ofisomers in the product mixture. The conditions of each reaction havebeen tailored to produce a particular ratio of the two isomers for thatcase.

In contrast to some synthetic pathways, where a mixture of isomers isproduced, the isomers may first be prepared individually, and then mixedin predetermined proportions to achieve the desired ratio. This conceptis illustrated in FIG. 10, where the product of one stereoselectivepathway is enriched in one of the isomers such that the productcomprises greater than about 75 percent by weight of the (E) isomer, andthe product of the other stereoselective pathway is enriched in theother isomer such that this product comprises greater than about 75percent by weight of the (Z) isomer. These numbers are exemplary too,and the purity of the desired isomer resulting from a stereoselectivepathway may be greater than or equal to about 75 percent by weight inone embodiment. In other embodiments the desired isomer may comprisegreater than or equal to about 80, 85, 90, and 95 percent by weight,respectively.

After synthesizing the isomers individually, they may be mixed toachieve the desired ratio, as illustrated in FIG. 10. For illustrativepurposes, the same hypothetical ratios are chosen in FIG. 10 as thoseused in FIGS. 9A–C. Referring to FIG. 10, the (E) and (Z)-isomers aremixed to yield three different mixtures that comprise ratios of the (E)to the (Z)-isomer of approximately 65 to 35 percent by weight, 50 to 50percent by weight, and 35 to 65 percent by weight, respectively.

In an alternative embodiment, a mixture of the (E) and (Z)-isomers ofISA_(TX)247 isomers may be separated such that the mixture is enrichedin one isomer over the other. For example, a Diels-Alder reaction may beused to convert the cis-isomer to a closed ring compound by reacting itwith an alkene. If the alkene is bound to a substrate that is capable ofisolation (e.g., filterable), the cis isomer may be substantiallyremoved from the mixture, leaving a composition enriched in the transisomer. The cis isomer may be re-constituted from the closed ringcompound with the application of heat, producing a composition enrichedin the cis isomer. Thus, in this manner, the cis and trans isomers maybe separated.

In practice, the ratio of the (E) to (Z)-isomers in any mixture,regardless of the degree of stereoselectively of the method by which itwas produced, may take on a broad range of values. For example, themixture may comprise from about 10 to 90 percent of the (E)-isomer toabout 90 to 10 percent of the (Z)-isomer. In other embodiments, themixture may contain from about 15 to 85 percent by weight of the(E)-isomer and about 85 to 15 percent of the (Z)-isomer; or about 25 to75 percent by weight of the (E)-isomer and about 75 to 25 percent byweight of the (Z)-isomer; or about 35 to 65 percent by weight of the(E)-isomer and about 65 to 35 percent by weight of the (Z)-isomer; orabout 45 to 55 percent by weight of the (E)-isomer and about 55 to 45percent of the (Z)-isomer. In still another embodiment, the isomericmixture is an ISA_(TX)247 mixture which comprises about 45 to 50 percentby weight of the (E)-isomer and about 50 to 55 percent by weight of the(Z)-isomer. These percentages by weight are based on the total weight ofthe composition, and it will be understood that the sum of the weightpercent of the (E) isomer and the (Z) isomer is 100 weight percent. Inother words, a mixture might contain 65 percent by weight of the(E)-isomer and 35 percent by weight of the (Z)-isomer, or vice versa.

The percentage of one isomer or another in a mixture can be verifiedusing nuclear magnetic resonance (NMR), or other techniques well knownin the art.

Pharmaceutical Compositions

This invention also relates to a method of treatment for patients inneed of immunosuppression involving the administration of pharmaceuticalcompositions comprising the inventive mixture as the activeconstituents. The indications for which this combination is of interestinclude in particular autoimmune and inflammatory conditions andconditions associated with or causal to transplant rejection, e.g.,treatment (including amelioration, reduction, elimination or cure ofetiology or symptoms) or prevention (including substantial or completerestriction, prophylaxis or avoidance) of the following:

-   -   a) Acute organ or tissue transplant rejection, e.g., treatment        of recipients of, e.g., heart, lung, combined heart-lung, liver,        kidney, pancreatic, skin, bowel, or corneal transplants,        especially prevention and/or treatment of T-cell mediated        rejection, as well as graft-versus-host disease, such as        following bone marrow transplantation.    -   b) Chronic rejection of a transplanted organ, in particular,        prevention of graft vessel disease, e.g., characterized by        stenosis of the arteries of the graft as a result of intima        thickening due to smooth muscle cell proliferation and        associated effects.    -   c) Xenograft rejection, including the acute, hyperacute or        chronic rejection of an organ occurring when the organ donor is        of a different species from the recipient, most especially        rejection mediated by B-cells or antibody-mediated rejection.    -   d) Autoimmune disease and inflammatory conditions, in particular        inflammatory conditions with an etiology including an        immunological or autoimmune component such as arthritis (for        example rheumatoid arthritis, arthritis chronica progrediente        and arthritis deformans) and other rheumatic diseases. Specific        autoimmune diseases for which the synergistic combination of the        invention may be employed include, autoimmune hematological        disorders (including e.g. hemolytic anemia, aplastic anemia,        pure red cell anemia and idiopathic thrombocytopenia), systemic        lupus erythematosus, polychondritis, sclerodoma, Wegener        granulomatosis, dermatomyositis, chronic active hepatitis,        myasthenia gravis, psoriasis, Steven-Johnson syndrome,        idiopathic sprue, (autoimmune) inflammatory bowel disease        (including e.g. ulcerative colitis and Crohn's disease),        endocrine ophthalmopathy, Graves disease, sarcoidosis, multiple        sclerosis, primary biliary cirrhosis, juvenile diabetes        (diabetes mellitus type I), uveitis (anterior and posterior),        keratoconjunctivitis sicca and vernal keratoconjunctivitis,        interstitial lung fibrosis, psoriatic arthritis,        glomerulonephritis (with and without nephrotic syndrome, e.g.        including idiopathic nephrotic syndrome or minimal change        nephropathy) and juvenile dermatomyositis. Autoimmune and        inflammatory conditions of the skin are also considered to be        amenable to treatment and prevention using the synergistic        combination of the invention, e.g., psoriasis, contact        dermatitis, atopic dermatitis, alopecia areata, erythema        multiforma, dermatitis herpetiformis, scleroderma, vitiligo,        hypersensitivity angiitis, urticaria, bullous pemphigoid, lupus        erythematosus, pemphigus, epidermolysis bullosa acquisita, and        other inflammatory or allergic conditions of the skin, as are        inflammatory conditions of the lungs and airways including        asthma, allergies, and pneumoconiosis.

The isomeric analogue mixtures of this invention may be administeredneat or with a pharmaceutical carrier to a warm-blooded animal in needthereof. The pharmaceutical carrier may be solid or liquid. Theinventive mixture may be administered orally, topically, parenterally,by inhalation spray or rectally in dosage unit formulations containingconventional non-toxic pharmaceutically acceptable carriers, adjuvantsand vehicles. The term parenteral, as used herein, includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection orinfusion techniques.

The pharmaceutical compositions containing the inventive mixture maypreferably be in a form suitable for oral use, for example, as tablets,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, or syrups or elixirs.Compositions intended for oral use may be prepared according to methodsknown to the art for the manufacture of pharmaceutical compositions andsuch compositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparation. Tablets containing the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients may alsobe manufactured by known methods. The excipients used may be forexample, (1) inert diluents such as calcium carbonate, lactose, calciumphosphate or sodium phosphate; (2) granulating and disintegrating agentssuch as corn starch, or alginic acid; (3) binding agents such as starch,gelatin or acacia, and (4) lubricating agents such as magnesiumstearate, stearic acid or talc. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated by the techniques described in the U.S. Pat. Nos. 4,256,108;4,160,452; and 4,265,874 to form osmotic therapeutic tablets forcontrolled release.

In some cases, formulations for oral use may be in the form of hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin. They may also be in the form of soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions normally contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients may include: (1) suspending agents such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; or(2) dispersing or wetting agents which may be a naturally-occurringphosphatide such as lecithin, a condensation product of an alkyleneoxide with a fatty acid, for example, polyoxyethylene stearate, acondensation product of ethylene oxide with a long chain aliphaticalcohol, for example, heptadecaethyleneoxycetanol, a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand a hexitol such as polyoxyethylene sorbitol monooleate, or acondensation product of ethylene oxide with a partial ester derived froma fatty acid and a hexitol anhydride, for example polyoxyethylenesorbitan monooleate.

The aqueous suspensions may also contain one or more preservatives, forexample, ethyl or n-propyl p-hydroxybenzoate; one or more coloringagents; one or more flavoring agents; and one or more sweetening agentssuch as sucrose, aspartame or saccharin.

Oily suspension may be formulated by suspending the active ingredient ina vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, a fish oil which contains omega 3 fatty acid, or in amineral oil such as liquid paraffin. The oily suspensions may contain athickening agent, for example beeswax, hard paraffin or cetyl alcohol.Sweetening agents and flavoring agents may be added to provide apalatable oral preparation. These compositions may be preserved by theaddition of an antioxidant such as ascorbic acid.

Dispersible powders and granules are suitable for the preparation of anaqueous suspension. They provide the active ingredient in a mixture witha dispersing or wetting agent, a suspending agent and one or morepreservatives. Suitable dispersing or wetting agents and suspendingagents are exemplified by those already mentioned above. Additionalexcipients, for example, those sweetening, flavoring and coloring agentsdescribed above may also be present.

The pharmaceutical compositions containing the inventive mixture mayalso be in the form of oil-in-water emulsions. The oily phase may be avegetable oil such as olive oil or arachis oils, or a mineral oil suchas liquid paraffin or a mixture thereof. Suitable emulsifying agents maybe (1) naturally-occurring gums such as gum acacia and gum tragacanth,(2) naturally-occurring phosphatides such as soy bean and lecithin, (3)esters or partial ester 30 derived from fatty acids and hexitolanhydrides, for example, sorbitan monooleate, (4) condensation productsof said partial esters with ethylene oxide, for example, polyoxyethylenesorbitan monooleate. The emulsions may also contain sweetening andflavoring agents.

Syrups and elixirs may be formulated with sweetening agents, forexample, glycerol, propylene glycol, sorbitol, aspartame or sucrose.Such formulations may also contain a demulcent, a preservative andflavoring and coloring agents.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to known methods using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or di-glycerides. In addition, fatty acidssuch as oleic acid find use in the preparation of injectables.

The inventive mixture may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials are cocoa butter and polyethylene glycols.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the disclosed cyclosporines are employed.

In a particularly preferred embodiment, a liquid solution containing asurfactant, ethanol, a lipophilic and/or an ampiphilic solvent asnon-active ingredients is used. Specifically, an oral multiple emulsionformula containing the isomeric analogue mixture and the followingnon-medicinal ingredients: d-alpha Tocopheryl polyethylene glycol 1000succinate (vitamin E TPGS), medium chain triglyceride (MCT) oil, Tween40, and ethanol is used. A soft gelatin capsule (comprising gelatin,glycerin, water, and sorbitol) containing the isomeric analogue mixtureand the same non-medicinal ingredients as the oral solution may alsopreferably be used.

Dosage levels of the order from about 0.05 mg to about 50 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions. The dose level and schedule ofadministration may vary depending on the particular isomeric mixtureused, the condition to be treated, and such additional factors as theage and condition of the subject. Preferred doses are from about 0.5 toabout 10 mg/kg/day and from about 0.1 to about 10 mg/kg/day. In apreferred embodiment, from about 2 to about 6 mg/kg/day is administeredorally b.i.d. In a particularly preferred embodiment, about 0.5 to about3 mg/kg/day is administered orally b.i.d.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, aformulation intended for the oral administration to humans may containfrom 2.5 mg to 2.5 g of active agent compounded with an appropriate andconvenient amount of carrier material which may vary from about 5 toabout 95 percent of the total composition. Unit dosage forms willgenerally contain between from about 5 mg to about 500 mg of activeingredient. In a preferred embodiment, individual capsules containingabout 50 mg isomeric mixture are employed for oral administration. Inanother preferred embodiment, oral solutions containing about 50 mg/mLisomeric mixture are used for oral administration.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination and the nature andseverity of the particular disease or condition undergoing therapy.

Methodology

The use of cyclosporine derivatives, a class of cyclic polypeptidesproduced by the fungus Tolypocladium inflatum Gams, is increasing inimmunosuppressive therapy due to their preferential effects on T-cellmediated reactions. Cyclosporine derivatives have been observed toreversibly inhibit immunocompetent lymphocytes, particularlyT-lymphocytes, as well as inhibit lymphokine production and release.This action is primarily mediated through cyclosporine A-inducedinhibition of calcineurin, a phosphatase enzyme found in the cytoplasmof cells (Schreiber and Crabtree, 1992). An indicator of the efficacy ofcyclosporine A or a cyclosporine A derivative is its ability to inhibitthe phosphatase activity of calcineurin. The calcineurin inhibitionassay measures the activity of the drug at its site of action, and, assuch, is the most accurate and direct in vitro assessment of the potencyof cyclosporine A analogues (Fruman et al., 1992).

ISA_(TX)247 is a cyclosporine A analogue that is similar to cyclosporineA, except for a novel modification of a functional group on the aminoacid 1 residue of the molecule. We have now found that ISA_(TX)247exhibits up to 3-fold greater potency than cyclosporine A in the invitro calcineurin inhibition assay.

Pharmacodynamic studies (in vivo and in vitro) have shown thatISA_(TX)247 has more potency than other existing cyclosporine compounds.The efficacy of isomeric mixtures of cyclosporine analogues ranging fromabout 10:90 to about 90:10 (trans- to cis-), in particular ISA_(TX)247having 50–55% Z-isomer and 45–50% E-isomer, as an immunosuppressiveagent (versus cyclosporine A) has been demonstrated in an in vitrocalcineurin activity assay, a rat heart transplant model, an islet cellallotransplantation mouse model, a collagen-induced arthritis model inthe mouse, and/or an antigen-induced arthritis model in the rabbit. Thedata show that these isomeric mixtures are equivalent to or more potentthan cyclosporine A, and therefore useful for the treatment ofimmunoregulatory disorders.

There are numerous adverse effects associated with cyclosporine Atherapy, including nephrotoxicity, hepatotoxicity, cataractogenesis,hirsutism, parathesis, and gingival hyperplasia to name a few (Sketriset al., 1995). Of these, nephrotoxicity is one of the more seriousdose-related adverse effects resulting from cyclosporine Aadministration. The exact mechanism by which cyclosporine A causes renalinjury is not known. However, it is proposed that an increase in thelevels of vasoconstrictive substances in the kidney leads to the localvasoconstriction of the afferent glomerular arterioles. This can resultin ischemia, a decrease in glomerular filtration rate, and over the longterm, interstitial fibrosis.

The nonclinical safety of ISA_(TX)247 has been evaluated in a number ofanimal species. Repeated-dose oral toxicity studies in rats, dogs, andprimates showed that ISA_(TX)247 was well-tolerated and produced effectsthat were consistent with immunosuppression. The only toxicologicaleffect noted in all species was diarrhea/loose feces.

ISA_(TX)247 does not exhibit mutagenic activity as demonstrated in invitro bacterial reverse mutation and chromosome aberration assays, andin an in vivo rat micronucleus assay. No carcinogenicity studies havebeen completed to date. Reproductive toxicity studies with ISA_(TX)247have been completed in pregnant rats and rabbits. There were notreatment-related malformations or alterations. At doses that resultedin maternal toxicity, corresponding embryotoxicity was observed.

EXAMPLES Example 1 Acetylation of Cyclosporine A

Acetic anhydride (140 milliliters) was added to Cyclosporin A (50.0grams, 41.6 millimoles) and the mixture stirred at room temperatureunder a N₂ atmosphere until all of the Cyclosporin A has dissolved.Dimethylaminopyridine (7.62 g, 62.4 mmol) was added and the reactionstirred at room temperature under a N₂ atmosphere for 3 hours or untilthe reaction was complete. The reaction mixture was cooled to 5° C. andthen filtered. The collected solids were washed with hexane to drive offadditional acetic anhydride. The resulting pasty solid was slowlytransferred to a vigorously stirred 5% aqueous sodium bicarbonatesolution (1.5 liters). The resulting suspension was stirred until a fineslurry was obtained and the evolution of CO₂ had ceased. The solids werecollected by filtration and washed with water until the filtrate hadneutral pH. The solid product was dried in a vacuum oven overnight (55°C.) to give 44.0 g (85%) of the product as a colorless solid.

Example 2 Oxidation of Product from Example 1

Acetonitrile (320 mL) and water (80 mL) were added to acetyl CyclosporinA (42.97 g, 34.54 mmol) and the mixture stirred until all of thematerial was dissolved. Sodium periodate (14.77 g, 69.08 mmol) wasadded, followed by the addition of ruthenium chloride hydrate (0.358 g,1.73 mmol) and then the reaction stirred at room temperature for 3 hoursunder a N₂ atmosphere. Water (300 mL) was added and the mixturetransferred to a separatory funnel. The mixture was extracted twice withethyl acetate (300 mL and then 250 mL). The dark black ethyl acetateextracts were combined and washed with 250 mL water followed by 250 mLbrine. The organic solution was then dried over MgSO₄ and the solventevaporated to give a greenish-black solid. The crude product waschromatographed over silica gel using 40% acetone/60% hexane as eluentto give the product (29.1 g, 68%) as a colorless solid.

Example 3 Preparation of Acetyl ISA_(TX)247

i) In situ Generation of ylide:

Acetyl Cyclosporin A aldehyde (31.84 g, 25.84 mmol) was added to 340 mLtoluene and the mixture stirred until the material was completelydissolved. To the resulting solution was added 340 mL of 1 normalaqueous sodium hydroxide. The resulting mixture was stirred vigorouslyand then allyl triphenylphosphonium bromide (58.22 g, 151.90 mmol)added. The reaction was stirred for 24 hours at room temperature andthen additional allyl triphenylphosphonium bromide (16.64 g, 43.42 mmol)added and stirring continued for a further 24 hours. The mixture wastransferred to a separatory funnel and the toluene phase separated. Theaqueous phase was extracted with an additional 200 mL of toluene. Thetwo toluene extracts were combined and washed sequentially with 200 mLdeionized water and 200 mL saturated aqueous sodium chloride solution.The solution was dried over MgSO₄, filtered, and the toluene evaporatedto give a very viscous gel. This material was treated with 142 mL ofethyl acetate and stirred until a fine slurry formed. Hexane (570 mL)was slowly added with rapid stirring. The stirring was continued for 30minutes and then the resulting suspension was filtered and the collectedsolids washed with 160 mL of 5:1 hexane/ethyl acetate. The combinedfiltrate was concentrated on a rotary evaporator to a viscoussemi-solid. This material was treated with 75 mL ethyl acetate andstirred until a fine slurry was obtained. Hexane (225 mL) was slowlyadded with rapid stirring. Stirring was continued for 30 minutes andthen the resulting suspension was filtered and the collected solidswashed with 100 mL of 5:1 hexane/ethyl acetate. The filtrate wasconcentrated on a rotary evaporator to give a pale yellow solid. Thecrude product was chromatographed over silica gel using 40% acetone/60%hexane as eluent to give the product (14.09 g) as a colorless solid.

ii) Pre-formed ylide Generation and Reaction in Presence of LiBr:

To a stirred suspension of allyltriphenyl phosphonium bromide (7.67 g,20 mmol) in THF (20 mL) being cooled to 0° C., was added a solution ofKOBu^(t) in tetrahydrofuran (20 mL, 20 mmol, 1 M solution). Stirring wascontinued at this temperature for 30 minutes and a solution of LiBr inTHF (10 mL, 10 mmol, 1 M solution) was added. The reaction mixture wasthen stirred for 30 minutes and a solution of acetyl CsA-aldehyde (4.93g, 4 mmol) in THF (10 mL) was added through a cannula. After stirringfor 15 minutes at room temperature, the reaction mixture was quenchedwith saturated NH₄Cl solution (25 mL). Workup and chromatography asabove furnished acetylated ISA_(TX)247 as a colorless solid (3.5 g).

Example 4 Preparation of ISA_(TX)247

Acetyl ISA_(TX)247 (14.6 g, 11.62 mmol) was dissolved in 340 mL ofmethanol and then 135 mL deionized water added. Potassium carbonate(13.36 g, 96.66 mmol) was added and the mixture stirred at roomtemperature for 24 to 48 hours until the reaction was complete. Most ofthe methanol was evaporated and then 250 mL ethyl acetate was added withstirring. A 10% aqueous citric acid solution (120 mL) was slowly addedand then the ethyl acetate phase separated. The aqueous phase wasextracted with an additional 200 mL portion of ethyl acetate. Thecombined ethyl acetate extracts were washed sequentially with 150 mLdeionized water, 100 mL 10% aqueous citric acid solution and 150 mLsaturated aqueous sodium chloride and then dried over MgSO₄. The ethylacetate was evaporated to give a pale yellow solid. The crude productwas chromatographed over silica gel using 40% acetone/60% hexane aseluent to give ISA_(TX)247 (10.51 g, 75%) as a colorless solid.ISA_(TX)247 contains 45–50% E-isomer and 50–55% Z-isomer.

The products in Examples 1–4 were characterized by mass spectrometryand/or nuclear magnetic resonance spectroscopy.

Example 5 Preparation of Acetyl-η-bromocyclosporin A

Acetyl Cyclosporin A (41.48 g, 33.3 mmol) prepared as in Example 1,N-bromosuccinimide (10.39 g, 58.4 mmol) and azo-bis-isobutyronitrile(1.09 g, 6.67 mmol) were dissolved in 250 mL of carbon tetrachloride andthe resulting mixture heated to reflux for 2.5 hours. The mixture wascooled and the solvent evaporated. The residue was treated with 350 mLdiethyl ether and filtered to remove the insoluble material. Thefiltrate was washed sequentially with 150 mL water and 150 mL brine,then dried over magnesium sulfate and the solvent evaporated. The crudematerial was chromatographed on silica gel with acetone/hexane (2:3) togive 28.57 g (65%) of acetyl-γ-bromocylosporin A as a yellow solid.

Example 6 Preparation of Triphenylphosphonium Bromide of AcetylCyclosporin A

Acetyl-γ-bromocylosporin A (28.55 g, 21.6 mmol) and triphenylphosphine(7.55 g, 28.8 mmol) were dissolved in 210 mL of toluene and theresulting solution heated to 100° C. for 21 hours. The solution wascooled and the toluene evaporated. The resulting oily, semi-solid wastreated with 250 mL of hexane/ether (1:4), mixed thoroughly and thesolvent decanted off. This process was repeated 3 more times with 150 mLether. The residue was then dissolved in 50 mL ethyl acetate andprecipitated with 220 mL hexane. The resulting solid was then collectedby filtration to give 22.5 g (66%) of triphenylphosphonium bromide ofacetyl cyclosporin A as a tan-colored solid.

Example 7 Wittig Reaction

The triphenylphosphonium bromide of acetyl cyclosporin (100 mg, 0.06mmol), an excess of 37% formaldehyde (0.25 mL) and toluene (2 mL) werestirred rapidly at room temperature. Aqueous sodium hydroxide as a 1Nsolution (2 mL) was added dropwise and stirring continued for 3.5 hours.The reaction mixture was diluted with ethyl acetate (20 mL) and water(10 mL). The ethyl acetate phase was separated, washed sequentially withwater (10 mL) and brine (10 mL), dried over magnesium sulfate and thesolvent evaporated. The crude material was chromatographed on silica gelwith acetone/hexane (2:3) to give 70 mg (88%) of a mixture of (E) and(Z)-isomers of acetyl ISA_(TX)247 as a colorless solid.

Example 8 De-acetylation of the Wittig Reaction Product

The mixture of isomers from Example 7 (70 mg., 0.056 mmol) was dissolvedin methanol (5 mL) and then water (1 mL) added. Potassium carbonate (75mg) was added and the reaction stirred at room temperature for 19 hours.Most of the methanol was evaporated and 15 mL ethyl acetate added to theresidue followed by 10 mL of 10% aqueous citric acid. The ethyl acetatephase was separated and the aqueous phase extracted with an additional10 mL of ethyl acetate. The combined ethyl acetate extracts were washedsequentially with 10 mL water, 10 mL 10% aqueous citric acid and 10 mLbrine before drying over magnesium sulfate and evaporating the solvent.The crude material was chromatographed on silica gel with acetone/hexane(2:3) to give 37 mg (54%) of ISA_(TX)247 as a colorless solid containingabout 85% E-isomer and about 15% Z-isomer.

The products in Examples 5–8 were characterized by mass spectrometryand/or nuclear magnetic resonance spectrometry.

Example 9 Preparation of the Geometrical Isomers of ISA_(TX)247

The cis- and trans-isomers of ISA_(TX)247 may be independentlysynthesized using the following reaction scheme. The sequence involvesknown metalation of allyltrimethylsilane, the electrophilic capture by atrimethylborate, followed by the hydrolysis and then transesterificationto generate the intermediate trans-(trimethylsilyl)allylboronate ester.Allylboration of cyclosporine aldehyde furnished a boron intermediate,which is converted to the desired β-trimethylsilyl alcohol, bysequestration. The diastereoselectivity in the creation of new chiralcenters is not determined at this stage due to removal of these centersat a later stage. It should be noted that the relative stereochemistryof the two centers in the β-trimethylsilyl alcohol is anti in agreementwith expectations and is due to the trans double bond in thetrans-(trimethylsilyl) boronate precursor.

Base-promoted elimination (Hudrlick et al., 1975) of β-trimethylsilylalcohol furnished a composition enriched in acetyl-(Z)-1,3-diene whileacid-promoted elimination gave a composition enriched inacetyl-(E)-1,3-diene. Deprotection leads to the respective dienealcohols, the (Z) and (E)-isomers of ISA_(TX)247, respectively.

An alternate approach to dienes utilizes the allylphosphoranes.Metalation of allyldiphenylphosphine and then transmetalation withTi(OPr^(i))₄ gives the titanium intermediate. Allyltitanation followedby stereospecific elimination would generate a composition enriched inthe (Z)-diene.

On the other hand, when allyldiphenylphosphine oxide is subjected to asimilar sequence (FIG. 8), the E-isomer is predominantly (75%)generated.

i) Allylboration of Acetyl CsA-CHO:

The (E)-1-trimethylsilyl-1-propene-3-boronate was prepared in accordancewith previously reported methods (Ikeda et al., 1987). To a stirredsolution of (E)-1-trimethylsilyl-1-propene-3-boronate (0.2 g, 0.832mmol) in THF (3 mL) under nitrogen was added acetyl Cyclosporin Aaldehyde (1.026 g, 0.832 mmol). The reaction mixture was monitored byhigh performance liquid chromatography (C-8 column, reverse phase) andstirred for a total period of 7 days. Then triethanolamine (0.196 g, 1.3mmol) was added and stirring continued for a further period of 4 days.The β-trimethylsilyl alcohol was obtained by purification over a silicagel column. MS(ES) m/z 1368.9 (M+Na⁺).

To a suspension of KH (3.5 mg, 26.4 μmol, 30% mineral oil dispersionwashed with anhydrous hexanes) in anhydrous THF (1 mL) was addedβ-trimethylsilyl alcohol (10 mg, 7.4 micromole) and stirred at roomtemperature for 10 min. The reaction mixture was diluted with diethylether (10 mL) and then washed with saturated NaHCO₃ solution (2×5 mL).Drying (Na₂SO₄) and solvent removal furnished the enriched(Z)-acetyl-1,3-diene. MS (ES) m/z 1294.8 (M+K⁺).

ii) Allyltitanation of Acetyl CsA-CHO:

To a stirred and cooled (−78° C.) solution of allyldiphenylphosphine(0.54 g, 2.4 mmol) in anhydrous THF (8 mL) was added t-BuLi (1.42 mL,2.4 mmol, 1.7 M solution in pentane). The brick-red colored solution wasstirred for 15 min at this temperature and then at 0° C. for 30 min. Itwas then cooled again to −78° C. and added Ti(OPr^(i))₄ (0.71 mL, 2.4mmol). The brown colored solution was stirred at this temperature for 15minutes and then a solution of acetyl CsA-CHO (2.5 g, 2 mmol) in THF (10mL) was added through a cannula. The pale-yellow colored solution wasstirred for a further period of 30 minutes and then warmed to roomtemperature overnight. To the reaction mixture was added MeI (0.15 mL,2.4 mmol) at 0° C. Stirring was continued for 1 h at this temperatureand then at room temperature for 2 h. The reaction mixture was pouredinto ice-cold 1% HCl (100 mL). The aqueous layer was extracted withEtOAc (3×50 mL). The combined organic extract was washed with water(2×25 mL) and brine (25 mL). Removal of solvent gave a yellow solidwhich was chromatographed over a column of silica gel. Elution with 1:3acetone-hexanes mixture furnished the (Z)-enriched isomer of acetylISA_(TX)247. Deprotection as in Example 4 gave (Z)-enriched isomer ofISA_(TX)247 (Z/E ratio, 75:25).

Example 10 Preparation of an (E)-enriched Mixture of ISA_(TX)247 Isomers

To a solution of allyldiphenylphosphine oxide (1 mmol) andhexamethylphosphoramide (2 mmol) in tetrahydrofuran (5 mL) at −78° C.was added n-butyllithium (1 mmol, in hexanes). The mixture was stirredat −78° C. for 30 minutes. A solution of acetyl cyclosporin A aldehyde(0.8 mmol) in tetrahydrofuran (7 mL) was added and the reaction mixtureallowed to gradually warm to room temperature and then stirred for 18hours. The mixture was poured into ice-cold 1N hydrochloric acid (50 mL)and then extracted into ethyl acetate. The organic extract was washedwith water, dried over magnesium sulfate and the solvent evaporated. Theresidue was chromatographed over silica gel using 25% acetone/75%hexanes as eluent to give a mixture of the (E) and (Z)-isomers of acetylISA_(TX)247. Removal of the acetate protecting group as described inExample 4 gave an (E)-enriched mixture of the ISA_(TX)247 isomers.Proton nmr spectroscopy indicated that the mixture was comprised of 75%of the (E) and 25% of the (Z)-isomer of ISA_(TX)247. This reaction wasalso carried out according to Schlosser's modification (R. Liu, M.Schlosser, Synlett, 1996, 1195). To a stirred and cooled (−78° C.)solution of allyldiphenylphosphine oxide (1.21 g, 5 mmol) in THF (20 mL)was added n-BuLi (2 mL, 5 mmol, 2.5 M solution in hexanes). Thered-colored solution was stirred for 40 minutes at −78° C. A solution ofacetyl CsA-CHO (1.25 g, 1.02 mmol) in THF (12 mL) was then added througha cannula during 15 minutes. The reaction mixture was stirred at roomtemperature for 2 hours. Workup and chromatography as above gave acetylISA_(TX)247 (Z:E ratio, 40:60 by ¹H NMR analysis).

Example 11 Preparation of Benzoyl-Protected Cyclosporin A

Cyclosporin A (6.01 g, 5 mmol) and 4-dimethylaminopyridine (305 mg, 2.5mmol) were dissolved in pyridine (5 mL). Benzoic anhydride (3.4 g, 15mmol) was added and the mixture stirred for 19 hours at 50° C.Additional benzoic anhydride (1.7 g, 7.5 mmol) and DMAP (305 mg, 2.5mmol) were added and stirring at 50° C. continued for another 24 hours.Benzoic anhydride (0.85 g, 3.8 mmol) was added and the reaction stirredfor an additional 23 hours. The reaction mixture was then poured slowlyinto water with stirring. Precipitated Cyclosporin A benzoate wasfiltered off and washed with water. The collected cake was dissolved ina minimum volume of methanol and added to a 10% citric acid solution andstirred for 1 hour. The precipitated product was collected by filtrationand washed with water until the pH of the filtrate reached that of thewater. The solid Cyclosporin A benzoate was dried at 50° C. under vacuumto give a colorless solid.

Example 12 Preparation of Triethylsilyl Ether-Protected Cyclosporin A

Cyclosporin A (3.606 g, 3 mmol) was dissolved in dry pyridine (8 mL) andthen DMAP (122 mg, 1 mmol) was added. The reaction mixture was cooled to0° C. and then triethylsilyl trifluoromethanesulfonate (3.6 mmol) addeddropwise. The mixture was allowed to warm to room temperature andstirred overnight. The reaction mixture was then poured slowly intowater with stirring. The precipitated triethylsilyl ether was filteredoff and washed with water. The collected cake was dissolved in a minimumvolume of methanol and added to a 5% citric acid solution and stirredfor 30 minutes. The precipitated product was collected by filtration andwashed with water until the pH of the filtrate reached that of thewater. The solid triethylsilyl ether was dried at 50° C. under vacuum togive a colorless solid. Triisopropylsilyl and tert-butyldimethylsilylprotecting groups were also introduced by following an analogousprocedure.

Example 13 Immunosuppressive Activity Using the Calcineurin InhibitionAssay

An indicator of the efficacy of cyclosporine A or a cyclosporine Aderivative is its ability to inhibit the phosphatase activity ofcalcineurin. The calcineurin inhibition assay measures the activity ofthe drug at its site of action and as such is the direct in vitroassessment of the potency of cyclosporine A analogues (Fruman et al.,1992).

The immunosuppressive activity of ISA_(TX)247 (45–50% of E-isomer and50–55% of Z-isomer) versus cyclosporine A has been assessed using thecalcineurin (CN) inhibition assay. The results of this assay show thatthe inhibition of calcineurin phosphatase activity by ISA_(TX)247(45–50% of Z-isomer and 50–55% of E-isomer) was up to a 3-fold morepotent (as determined by IC₅₀) as compared to cyclosporine A (FIG. 11).

The immunosuppressive activity of various deuterated and non-deuteratedisomeric analogue mixtures versus cyclosporine A has been assessed usingthe calcineurin (CN) inhibition assay. The structure and isomericcomposition of these analogues is set forth in FIG. 12. In FIG. 12, thedesignation “I4” corresponds to the structure of ISA_(TX)247. I4-M2denotes ISA_(TX)247 produced by the method described in Examples 5–8(designated Method 2 in this figure). I4-D4 denotes deuteratedISA_(TX)247 produced by the method described in Examples 1–4. I4-D2denotes deuterated ISA_(TX)247 produced by the method described inExamples 5–8. Other isomeric mixtures are as shown in the figure.

The results of this assay show that the inhibition of calcineurinphosphatase activity by these isomeric analogue mixtures was at least aspotent (as determined by IC₅₀) as compared to cyclosporine A (FIG. 13).CsA denotes Cyclosporine A; Isocyclo4 denotes ISA_(TX)247 produced bythe method described in Examples 1–4. Isocyclo5 corresponds to I5-M1 ofFIG. 12. Isocyclo4-d4 corresponds to I4-D4 of FIG. 12. Isocyclo5-d5corresponds to I5-D5 of FIG. 12. Isocyclo4-d2 corresponds to I4-D2 ofFIG. 12. Isocyclo4-M2 corresponds to I4-M2 of FIG. 12. Isocyclo5-m2corresponds to I5-M5 of FIG. 12.

Example 14 Immunosuppressive Activity Using the Rat Heart TransplantModel

The efficacy of ISA_(TX)247 (45–50% of E-isomer and 50–55% of Z-isomer)in preventing the rejection of hearts transplanted between differentstrains of rats was assessed and compared to that of cyclosporine A. Therat heart transplant model has been the most frequently used model toassess the in vivo potency of new immunosuppressive drugs, as prolongedgraft survival is difficult to achieve in this model due to immunerejection.

The procedure involved the heterotopic transplantation (to the abdominalaorta and inferior vena cava) of the heart from Wistar Furth rats toLewis rats. Intraperitoneal injections of either cyclosporine A or anisomeric analogue mixture were given to the transplant recipientstarting 3 days prior to transplantation and continuing for 30 dayspost-transplantation. If graft dysfunction was noted during the 30-daypost-transplantation period, the animal was sacrificed. If the animalsurvived longer than 30 days post-transplantation, the test and controlarticles were discontinued and the animal was allowed to continue untilgraft dysfunction or up to 100 days post-transplantation.

The average survival rates for each group of recipient animals aresummarized in Table 1. These results show that ISA_(TX)247 (45–50% ofE-isomer and 50–55% of Z-isomer) at an optimal dose of 1.75 mg/kg/dayincreased survival time approximately 3-fold over Cyclosporine A. Anumber of animals receiving ISA_(TX)247 still had functioning grafts at100 days post-transplant (70 days post discontinuation of dosing). Thesedata demonstrate the immunosuppressive activity of this isomericanalogue mixture in preventing graft rejection.

TABLE 1 Effect of ISA_(TX)247 and Cyclosporine A Given byIntraperitoneal Administration on the Average Survival Times ofTransplanted Rat Hearts [averaged from two separate studies, n 13]Average Survival Time (days post-operative) Dose Mean ± SEM (scanningelectron microscope) (mg/kilogram/day) Vehicle Control Cyclosporine AISA_(TX)247 0 9 ± 1 0.5 13^(a) ± 4 11^(a) ± 2  1.75 18^(b) ± 7 57^(b) ±32 3 50^(c) ± 8 55^(c) ± 12 ^(a,c)Not significantly different^(b)Significantly different (p < 0.01)

The efficacy of various deuterated and non-deuterated isomeric analoguemixtures (structures given in FIG. 12) in preventing the rejection ofhearts transplanted between different strains of rats was also assessedand compared to that of cyclosporine A. Doses were at 1.75 mg/kg/day for30 days. Results are summarized in Table 2. These results show that theisomeric mixtures at 1.75 mg/kg/day increased survival time at least asmuch as Cyclosporine A and demonstrate the immunosuppressive activity ofthese isomeric analogue mixtures in preventing graft rejection.

TABLE 2 Effect of Various Isomeric Analogue Mixtures and Cyclosporine AGiven by Intraperitoneal Administration at 1.75 mg/kg/day on the AverageSurvival Times of Transplanted Rat Hearts Average Survival Time (dayspost- Test Compound operative) Vehicle Control  9 Cyclosporine A 20I5-M1 20 I4-M2   20+ I4-D2 30

Example 15 Immunosuppressive Activity in Islet Cell Allotransplantation

The ability of ISA_(TX)247 (45–50% of E-isomer and 50–55% of Z-isomer)versus cyclosporine A to prolong the survival of transplanted isletcells in a mouse model was investigated in a study involving thetransplant of 500 islets from a CBA/J mouse into the renal capsule ofdiabetic Balb/c mouse recipients.

Following transplantation, ISA_(TX)247 or cyclosporine A wasadministered by intraperitoneal (i.p.) injection at a dose level of 0(vehicle), 1.75, 10, 20, or 25 mg/kg/day for a total of 30 days. Bloodglucose was monitored daily until the time of graft failure, as definedby a glucose level greater than 17 mmol/L on two consecutive days.

The results indicate that ISA_(TX)247 increased the length of graftsurvival by 40% at a dose of 20 mg/kg/day (Table 3). It was also notedthat ISA_(TX)247 was less toxic than cyclosporine A as the dose levelincreased. This was especially apparent at the 25 mg/kg/day dose level.

TABLE 3 The Survival of Mouse Islet Allografts in Diabetic MiceReceiving Either ISA_(TX)247 or Cyclosporine A by IntraperitonealInjection at a Dose Level of 1.75, 10, 20, or 25 mg/kg/day Dose MedianMean (mg/kg/day) Treatment N Survival Survival 0 Vehicle 7 17 16.8 1.75CsA 9 17 17.4 1.75 ISA 9 18 18.7 10 CsA 6 21 25.3 10 ISA 5 18 19.2 0Vehicle 12 16 15.9 20 CsA 9 19 20.2 20 ISA 9 >28 >28 0 Vehicle 5 21 21.125 CsA 10 ND* ND* 25 ISA 8 50 46.4 *7 out of the 10 animals in thisgroup died of CsA toxicity. Therefore, only 3 animals completed in thisgroup and no statistics were done.

Example 16 Immunosuppressive Activity in Arthritis

Over the course of the past three decades, three animal models of humanrheumatoid arthritis have been extensively examined and widely employedin the preclinical screening and development for novel anti-rheumaticagents. These include the adjuvant-induced, collagen-induced, andantigen-induced arthritis models. The following studies were designed toevaluate anti-inflammatory efficacy of ISA_(TX)247 (45–50% of E-isomerand 50–55% of Z-isomer) in both the collagen-induced arthritis model inthe mouse and the antigen-induced arthritis model in the rabbit. Thehistopathology and immunopathology observed in these two models resemblethe findings in the human disease. In both models, the efficacy ofISA_(TX)247 to prevent the onset of arthritis (prevention protocol) andto treat arthritis (treatment protocol) was examined. These studiessupport the immunosuppressive action of the claimed isomeric analoguemixtures.

A. Collagen-Induced Arthritis

Male DBA/1 Lac J mice, kept under virus antibody free conditions, wereimmunized subcutaneously at 8 to 10 weeks of age with 100 microgram ofchick type II collagen, emulsified in Freund's complete adjuvant.ISA_(TX)247, cyclosporine A, or vehicle (Chremophor EL/ethanol 72:28,volume/volume) were administered daily by intraperitoneal (i.p.)injection of 1- to 50-fold dilutions of stock drug (0.25, 0.5, or 1mg/mL) into saline to yield concentrations of 0 (vehicle); 125, 250, or500 μg/mouse for ISA_(TX)247; and 250, or 500 μg/mouse for cyclosporineA. Animals assigned to the prevention protocol (12/group) were dosedstarting on the day of immunization with collagen (Day 0) untilsacrifice on Day 40. Animals assigned to the treatment protocol(12/group) were dosed starting on the day of disease onset (˜Day 28)until sacrifice on Day 38.

Evaluated parameters included mortality, serum creatinine, histology,and outcome assessments, such as clinical scoring (visual), hind pawswelling, histological scoring, erosion scoring, andimmunohistochemistry.

Erosion scoring was done in a blinded manner by examining sagittalsections of the proximal interphalangeal (PIP) joint of the middle digitfor the presence or absence of erosions (defined as demarcated defectsin cartilage or bone filled with inflammatory tissue). This approachallowed for comparisons of the same joint. Previous studies havedemonstrated erosions in >90% of untreated arthritic animals in thisjoint.

The results indicate that the negative erosion scores in the ISA_(TX)247high-dose treatment group (500 μg/mouse) were significantly higher thanthe negative erosion scores in the vehicle treatment group (p<0.05).Both the mid-dose ISA_(TX)247 (250 μg/mouse) and high-dose cyclosporineA (500 μg/mouse) treatment groups had higher negative erosion scores ascompared to the vehicle treatment group (p<0.1). Furthermore, thelow-dose ISA_(TX)247 (125 μg/mouse) and mid-dose cyclosporine A control(250 μg/mouse) treatment groups have higher, although not statisticallysignificant, negative erosion scores when compared to the vehiclecontrol group.

The only treatment to significantly prevent the development of jointerosions was ISA_(TX)247 at 500 μg/mouse. This significant reduction inthe proportion of the PIP joints showing erosive changes in theISA_(TX)247-treated mice relative to the vehicle control group micedemonstrates that ISA_(TX)247 has disease-modifying properties.

B. Antigen-Induced Arthritis

New Zealand White rabbits, maintained under specific pathogen freeconditions, were immunized with 10 mg of ovalbumin in saline emulsifiedwith Freund's complete adjuvant that was given intramuscularly andsubcutaneously into several sites in the nape of the neck. Fourteen dayslater, all animals started receiving 2 daily intra-articular injectionsof 5 mg ovalbumin and 65 ng of human recombinant transforming growthfactor 2 in saline.

ISA_(TX)247, cyclosporine A, or vehicle (Chremophor EL/ethanol 72:28,V/V) were administered daily by subcutaneous injection of 1- to 4-folddilutions of stock drug (in vehicle) into saline to yield concentrationsof 0 (vehicle); 2.5, 5.0, or 10 mg/kg/day for ISA_(TX)247; and 5.0, 10,or 15 mg/kg/day for cyclosporine A. Animals assigned to the preventionprotocol (8/group) were dosed starting on the day of immunization withovalbumin (Day 0) until sacrifice on Day 42. Animals assigned to thetreatment protocol (8/group) were dosed starting on the day of diseaseonset (˜Day 28) until sacrifice on Day 42.

Evaluated parameters included mortality, body weight, serum creatinine,histology, and outcome assessments such as knee joint swelling, synovialfluid counts, gross postmortem analysis, and histology.

A significant decrease in synovial histopathological scores was observedin ISA_(TX)247 (P 0.05) and cyclosporine A (P 0.05) animals after 28days of therapy (prevention protocol) compared to vehicle controlanimals. This was accompanied by significant reductions in synovialfluid counts (ISA_(TX)247, P 0.05; cyclosporine A, P 0.05). Significantamelioration in synovial histopathological scores of animals withestablished arthritis was also evident following 14 days of treatmentwith ISA_(TX)247 (P 0.05) and cyclosporine A (P 0.05) compared tovehicle controls (treatment protocol). A significant reduction inmacroscopic arthritis score was evident in ISA_(TX)247 (P=0.01), but notin cyclosporine A treated animals. Treatment was well tolerated with nosignificant toxicity upon analysis of serum creatinine or post-mortemhistology.

The data show that ISA_(TX)247 is equivalent or potentially more potentthan cyclosporine A in the treatment and prevention of rheumatoidarthritis in an antigen-induced arthritis model in the rabbit.

Example 17 Pharmacokinetic and Toxicokinetic Properties

The pharmacokinetic and toxicokinetic parameters of ISA_(TX)247 (45–50%of E-isomer and 50–55% of Z-isomer) and cyclosporine A were tested in arabbit model. The rabbit has also been used as a model to studycyclosporine A nephrotoxicity, but far less frequently than the rat.Studies have found that cyclosporine A administered to the rabbit causesstructural and functional changes at a dose not only lower than has beenpreviously reported in other animal models, but also within at least theupper level of the therapeutic range in humans (Thliveris et al., 1991,1994). Also, the finding of interstitial fibrosis and arteriolopathy, inaddition to the cytological changes in the tubules, suggests that therabbit is a more appropriate model to study nephrotoxicity, since thesestructural entities are hallmarks of nephrotoxicity observed in humans.ISA_(TX)247 was administered intravenously (i.v.) for the first 7 daysand subcutaneously (s.c.) for an additional 23 days according to thefollowing schedule.

TABLE 4 The Dose Administration Schedule for the Investigation of thePharmacokinetic and Toxicokinetic Properties of ISA_(TX)247 in theRabbit Model Days 1–7: Days 8–30: i.v. Dose s.c. Dose Number of AnimalsTreatment Group (mg/kg) (mg/kg) Males Females 1. Vehicle Control  0  0 44 2. Cyclosporine A 10 10 6 6 Control 3. Low-Dose  5  5 0 2 4.Medium-Dose 10 10 4 4 5. High-Dose 15 15 4 4

Pathogen free rabbits (SPF) were used to ensure any renal changesobserved were due to the effect of ISA_(TX)247 and not due to pathogens.On Days 1 and 7, blood samples were collected prior to drugadministration and at 0.5, 1, 2, 4, 8, 12, 18, and 24 hours post-dose togenerate a pharmacokinetic profile. Other evaluated parameters includedclinical observations, body weight, food consumption, hematology,clinical chemistry, gross pathology, and histopathological examinationof selected tissues/organs.

Blood samples were analyzed via high performance liquid chromatographycoupled with mass spectrometry (LCMS). Table 5 below summarizes theaverage pharmacokinetic parameters in rabbits that received 10 mg/kg ofcyclosporine A or ISA_(TX)247.

TABLE 5 Pharmacokinetic Parameters of Intravenously AdministeredCyclosporine A and ISA_(TX)247 in Male Rabbits Receiving 10 mg/kg/day.Results expressed as mean ± SD Measured Cyclosporine A ISA_(Tx)247Parameter Day 1 Day 7 Day 1 Day 7 t_(max) (hours) 0.5 0.5 0.5 0.5C_(max) (μg/L) 1954 ± 320  2171 ± 612  1915 ± 149  1959 ± 470  t½(hours) 7.4 ± 2.8 9.0 ± 4.0 7.4 ± 1.7 9.2 ± 1.1 Area under the 6697 ±1717 6685 ± 1247 5659 ± 1309 5697 ± 1373 curve (μg · hr/L)

There were no statistically significantly differences between thepharmacokinetic parameters of cyclosporine A and ISA_(TX)247 in malerabbits receiving 10 mg/kg/day. The pharmacokinetic parameters ofISA_(TX)247 in female rabbits receiving the same dose were notsignificantly different from that observed in the male rabbits, with theexception of maximum concentration on Day 7.

No significant changes were noted in the hematological parameters ofrabbits receiving a vehicle control, cyclosporine A, or ISA_(TX)247. Adifference was noted in the creatinine levels in the various groups overthe course of the study, as is shown in Table 6 below. These differencesindicated that cyclosporine A had a significantly greater negativeeffect on the kidneys than either the vehicle control or ISA_(TX)247. Itshould be noted that even at a 50% higher dose, 15 mg/kg/day, ascompared to 10 mg/kg/day cyclosporine A, ISA_(TX)247 did not result inany significant increase in serum creatinine levels.

TABLE 6 Percent Change in Serum Creatinine Levels Over Baseline in MaleRabbits Receiving Vehicle, Cyclosporine A, or ISA_(TX)247 for 30 DaysTreatment Group Day 15 Day 30 Vehicle  +6%  −3% Cyclosporine A (10mg/kg) +22% +33% ISA_(TX)247 (10 mg/kg)  +1% +10% ISA_(TX)247 (15 mg/kg)−19% −11%

Examination of organs in all rabbits receiving the vehicle control, 10mg/kg cyclosporine A, 5 mg/kg ISA_(TX)247, or 10 mg/kg ISA_(TX)247revealed no significant abnormalities. This was especially true for thekidneys, in which no evidence of interstitial fibrosis, normally seen incyclosporine A-treated animals (Thliveris et al., 1991, 1994) was noted.In male rabbits that received 15 mg/kg ISA_(TX)247, a decrease inspermatogenesis was noted. No changes were noted in the 3 female rabbitsthat completed the study at this dose of 15 mg/kg ISA_(TX)247.

Example 18 Immunosuppressive Effects of ISA_(TX)247

Whole blood from cynomolgous monkeys (n=4) was incubated withISA_(TX)247 or cyclosporine and stimulated with different mitogens inculture medium. Lymphocyte proliferation was assessed by tritium-labeledthymidine incorporation and by flow-cytometric analysis of expression ofproliferating cell nuclear antigen (PCNA) on cells in SG₂M phase. Flowcytometry was also used to assess production of intracellular cytokinesby T cells and expression of T lymphocyte activation antigens. The EC₅₀(concentration of drug necessary to attain 50% of the maximum effect)was subsequently calculated using the WinNonlin™ software. Resultsshowed that lymphocyte proliferation, cytokine production, andexpression of T cell surface antigens were inhibited more potently byISA_(TX)247 than by cyclosporine, as shown by the EC₅₀ (expressed inng/mL) set forth in Table 7 below.

TABLE 7 Parameter ISA_(TX)247 Cyclosporine ³H-thymidine uptake 160.54565.52 PCNA expression 197.72 453.88 IL - 2 production 103.35 504.80IFN - production 102.67 465.65 TNF - production  90.58 508.29 CD 71expression 149.84 486.82 CD 25 expression 121.00 431.53 CD 11aexpression 204.40 598.90 CD 95 expression 129.98 392.97 CD 154expression 160.87 975.10

Thus, using an ex vivo whole blood assay we have found that ISA_(TX)247suppresses diverse immune functions 2.3–6 times more potently thancyclosporine.

Example 19 Wittig Reaction Using Tributyl Allyl Phosphonium Bromide

Potassium tert butoxide (0.31 g, 2.8 mmol) was dissolved in 20 mL oftetrahydrofuran. At about −40° C. tributyl allyl phosphonium bromide(0.99 g, 3.1 mmol) dissolved in 3 mL of tetrahydrofuran was slowlyadded. The resulting yellow mixture was stirred for about 10 minutes atabout −40° C. before a solution of acetyl cyclosporin A aldehyde (1.5 g,1.2 mmol) in 6 mL of tetrahydrofuran was slowly added. After stirringthe yellow-orange reaction mixture for 1.5 hours the reaction wascomplete. For quenching the reaction mixture was transferred ontoaqueous phosphoric acid (1.2 g, 1.0 mmol). The resulting aqueoussolution was extracted with 100 mL of toluene followed by 50 mL oftoluene. The combined organic layers were washed with water andconcentrated under reduced pressure to dryness. The product, acetylatedISA_(TX)247, was obtained as a slightly yellow solid in approximately90% yield. The isomer ratio was about 87% E-isomer and about 13%Z-isomer (as determined by ¹H-NMR spectroscopy).

Example 20 Wittig Reaction Using Tributyl Allyl Phosphonium Bromide anda Lithium Base

Tributyl allyl phosphonium bromide (1.38 g, 4.3 mmol) was dissolved in amixture of 20 mL of toluene and 3 mL of tetrahydrofuran. At about −78°C. butyllithium (1.6 M in hexane, 2.43 mL, 3.9 mmol) was slowly added.The resulting yellow mixture was stirred for about 10 minutes at about−78° C. before a solution of acetyl cyclosporin A aldehyde (1.5 g, 1.2mmol) in 6 mL of toluene was slowly added. After stirring theyellow-orange reaction mixture for 3.5 hours the reaction was quenchedby transferring the reaction mixture onto a mixture of 50 mL toluene andaqueous phosphoric acid (0.25 g, 2.2 mmol). The resulting biphasicmixture was allowed to warm to ambient temperature before the two layerswere separated. The toluene layer was washed with 20 mL water andconcentrated under reduced pressure to dryness. The product, acetylatedISA_(TX)247, was obtained as a slightly yellow solid in approximately80% yield. The isomer ratio was about 70% E-isomer and about 30%Z-isomer (as determined by ¹H-NMR spectroscopy).

Example 21 Wittig Reaction Using Tributyl Allyl Phosphonium Bromide anda Lithium Base

Running SAP018 as described above but only at about −40° C. Theexperimental conditions of Example 20 were repeated, this time using areaction temperature of about −40° C. Under these conditions theisomeric ratio of the isolated product, acetylated ISA_(TX)247, wasabout 74% by weight of the E-isomer, and to about 26% by weight of theZ-isomer, as determined by ¹H-NMR-spectroscopy.

Example 22 Wittig Reaction Using Tributyl Allyl Phosphonium Bromide

A solution of acetyl cyclosporin A aldehyde (1.5 g, 1.2 mmol) andtributyl allyl phosphonium bromide (0.99 g, 3.1 mmol) in 15 mL oftetrahydrofuran was cooled to about −80° C. Potassium tert-butoxide(0.19 g, 1.7 mmol) dissolved in 9 mL of tetrahydrofuran was slowlyadded. The resulting yellow mixture was stirred for one hour at about−80° C. to complete the reaction before a solution of 6 mL oftetrahydrofuran was slowly added. After stirring the yellow-orangereaction mixture for 1.5 hours the reaction was complete. For quenchingthe reaction mixture aqueous phosphoric acid (0.15 g, 1.3 mmol) wasadded. The resulting mixture was concentrated and the residue wasdissolved in 5 mL of methanol. Then the mixture was slowing added to 5mL of water. The resulting precipitate was filtered, washed with 4 mL ofmethanol/water (1/1), and dried in vacuo. The product, acetylatedISA_(TX)247, was obtained as a colorless solid in approximately 90%yield. The isomer ratio was about 91% by weight E-isomer and 9% byweight Z-isomer (determined by ¹H-NMR-spectroscopy).

Example 23 Ozonolysis of Acetyl CsA

A solution of acetyl cyclosporin A (15 g, 12.1 mmol) in 200 mL ofmethanol was ozonised at −78° C. using a Sander ozone generator at about1.1 bar with a current flow of 300 L O₂/hour until the reaction wascomplete (about 5 minutes). The solution was gassed with argon andquenched with dimethylsulfide dissolved in methanol. For completing thereduction the mixture was stirred overnight at room temperature. Afterconcentration to about 50 mL the solution was slowly added to 500 mL ofwater. The resulting precipitate was filtered, washed with 60 mL ofwater and dried in vacuo. The product, acetylated CsA aldehyde, wasobtained as a colorless solid in approximately 95% yield and a purity ofabout 98% (determined by HPLC).

Example 24 Preparation of Trimethylsilyl-protected Cyclosporine A

Cyclosporine A (40 g, 1 equivalent) was dissolved in dichloromethane(100 ml) at 30° C. N,N-bis-(trimethylsilyl) urea (1.1 equivalent) wasadded. After 5 minutes stirring at 30° C., p-toluenesulfonic acid (0.02equivalents) was added. The reaction mixture was heated at reflux untilcompletion of the reaction, as measured by thin layer chromatography(TLC), high pressure or high performance liquid chromatography (HPLC) ormass spectroctrometry (MS) and then cooled to room temperature. Halfsaturated aqueous sodium bicarbonate solution (100 ml) was added. Theaqueous phase was separated and re-extracted with dichloromethane. Thecombined organic phases were dried over anhydrous Na₂SO₄ and filtered.The solvent was removed under reduced pressure providing the crudetrimethylsilyl-protected Cyclosporine A.

Example 25 Preparation of Trimethylsilyl-protected Cyclosporine AAldehyde

Trimethysilyl-protected Cyclosporine A (5 g, 1 equivalent) was dissolvedin dichloromethane (50 ml). The solution was then cooled to atemperature of about −78° C., after which ozone was bubbled through thesolution until the appearance of a blue color. Next, argon was bubbledthrough the solution until a colorless solution was obtained in order toremove excess ozone it became colorless; this step was carried out toremove excess ozone. Triethylamine (5 equivalents) was added and thereaction mixture was stirred at room temperature for 17 hours. Thetrimethylsilyl-protected Cyclosporine A aldehyde was obtained afteraqueous work-up.

Example 26 Preparation of a 3:1 Mixture of Z to E Double Bond Isomers ofTrimethylsilyl-protected Cyclosporine A Diene Via Wittig Reactions

To a mixture of potassium tert-butoxide (3 equivalents) andallyltriphenylphosphonium bromide (2 equivalents) in toluene (10 ml)previously stirred for 60 minutes, was added thetrimethylsilyl-protected Cyclosporine A aldehyde (1 g, 1 equivalent).Work-up of the reaction mixture after 1 hour reaction at roomtemperature provided a 3:1 mixture (by NMR) of Z and E double bondisomers of the trimethylsilyl-protected Cyclosporine A diene.

Example 27 Preparation of a 1:1 Mixture of Z to E Double Bond Isomers ofTrimethylsilyl-protected Cyclosporine A Diene Via Wittig Reactions

The trimethylsilyl-protected Cyclosporine A aldehyde (2.5 g) wasdissolved in 25 ml of toluene and treated with 1N aqueous sodiumhydroxide solution (10 equivalents). The reaction mixture was vigorouslystirred and allyltriphenylphosphonium bromide (7.5 equivalents,portionwise) was added. Work-up of the reaction mixture after severalhours reaction at room temperature provided a ca 1:1 mixture (by NMR) ofZ and E double bond isomers of the trimethylsilyl-protected CyclosporineA diene.

Example 28 Preparation of a 1:2 Mixture of Z to E Double Bond Isomers ofTrimethylsilyl-protected Cyclosporine A Diene Via Wittig Reactions

The trimethylsilyl-protected Cyclosporine A aldehyde (1 g) was dissolvedin 5 ml of toluene together with potassium carbonate (1.5 equivalent)and allyltriphenylphosphonium bromide (1.5 equivalent). Work-up of thereaction mixture after 4 hours reaction at reflux under vigorousstirring provided a ca 1:2 mixture (by (NMR) of Z and E double bondisomers of the trimethylsilyl-protected Cyclosporine A diene.

Example 29 Preparation of a 1:3 Mixture of Z to E Double Bond Isomers ofTrimethylsilyl-protected Cyclosporine A Diene Via Wittig Reactions

Allyltributylphosphonium bromide (3 equivalents, prepared fromallylbromide and tributylphosphine) was dissolved in THF (3.5 ml).Toluene (7.5 ml) was added followed by potassium tert-butoxide (4equivalents). After 1 hour stirring at room temperature, the solutionwas cooled to ca −30° C. A solution of the trimethylsilyl-protectedCyclosporine A aldehyde (1 g, 1 equivalent) in toluene (5 mL) was addeddropwise. After 45 minutes at about −30° C., the reaction mixture wasworked up, providing an approximately 1:3 mixture (by NMR) of Z and Edouble bond isomers of the trimethylsilyl-protected Cyclosporine Adiene.

The following two examples, Examples 30 and 31, are directed toallylmetallations.

Example 30 Preparation of Acetyl-protected Cyclosporine Aβ-trimethylsilylalcohol

To a solution of allyltrimethylsilane (10.1 equivalents) in THF (15 ml)was added butyl lithium (1.6 M in hexanes, 10 equivalents) at roomtemperature. After 30 minutes reaction, the solution was cooled to −75°C., and treated with diethyl-B-methoxyborane (10.1 equivalents). After 1hour, borontrifluoride diethylether complex (10.1 equivalents) was addedto generate the B-(γ-trimethylsilyl-allyl)-diethylborane reagent. After1 hour, a solution of acetyl-protected Cyclosporine A aldehyde (5 g, 1equivalent) in THF (15 ml) was added dropwise. After 20 minutes, thereaction mixture was warmed to −10° C. and a saturated aqueous NH₄Clsolution was added. After stirring one hour at room temperature, water(45 ml) was added and the reaction mixture was extracted 3 times with 25ml ethyl acetate. The organic phases were washed sequentially with water(25 ml) and a saturated aqueous NH₄Cl solution (25 ml). The combinedorganic phases were dried over Na₂SO₄, filtered, and concentrated underreduced pressure. The crude product was chromatographed (Silicagel,dichloromethane/methanol or ethyl acetate/heptane) to yield theacetyl-protected Cyclosporine A β-trimethylsilylalcohol.

Example 31 Preparation of Trimethylsilyl-protected Cyclosporine Aβ-Trimethylsilylalcohol

To a solution of allyltrimethylsilane (10.1 equivalent) in THF (15 ml),was added butyl lithium (1.6 M in hexanes, 10 equivalents) at roomtemperature. After allowing the reaction to proceed for about 30minutes, the solution was cooled to −65° C., and treated withdiethyl-B-methoxyborane (10.1 equivalents). After 1 hour,borontrifluoride diethylether complex (10.1 equivalents) was added togenerate the B-(γ-trimethylsilyl-allyl)-diethylborane reagent. After 1hour, a solution of trimethylsilyl-protected Cyclosporine A aldehyde (5g, 1 equivalent) in THF (15 ml) was added dropwise. After 15 minutes,the reaction mixture was warmed to 10° C. and a saturated aqueous NH₄Clsolution was added. After [1 hour] stirring for one hour at roomtemperature, water (12.5 ml) and saturated NaHCO₃ (25 ml) were added.The reaction mixture was extracted twice with 25 ml methyl-t-butylether. The organic phases were washed twice sequentially with water(2×25 ml) and a saturated aqueous NaCl solution (25 ml). The combinedorganic phases were dried over Na₂SO₄, filtered, and concentrated underreduced pressure. The crude product was chromatographed (Silicagel,heptane/ethyl acetate) to yield the trimethylsilyl-protectedCyclosporine A β-trimethylsilylalcohol.

The following three examples, Examples 32, 33, and 34, are directed toPeterson elimination reactions.

Example 32 Preparation of E-acetyl-protected Cyclosporine A Diene

The acetyl-protected Cyclosporine A β-trimethylsilylalcohol (10 g, 1equivalent) was dissolved in THF (50 ml). Concentrated H₂SO₄ (1.24 ml, 3equivalent) was added and the reaction mixture was stirred for 20 h atroom temperature. Water (150 ml) was added and the reaction mixture wasextracted with methyl-t-butyl ether (200 ml). The aqueous phase wasre-extracted with methyl-t-butyl ether (150 ml). The organic phases werewashed with water (150 ml). The combined organic phases were dried overNa₂SO₄, filtered and concentrated under reduced pressure to give thecrude acetyl-protected Cyclosporine A diene (acetyl-protectedISA_(TX)247). The crude product was crystallized from methyl-t-butylether/THF and then recrystallized from methyl-t-butyl ether/DCM to giveacetyl-protected Cyclosporine A diene (acetyl-protected ISA_(TX)247) asa 99–97%:1–3% mixture of E and Z double bond isomers (by 400 MH_(Z) NMR,2% error of measurement).

Hydrolysis of E-acetyl-protected Cyclosporine A diene was conducted asfollows: Acetyl Cyclosporine A diene (4 g, 1 equivalent) was dissolvedin methanol (80 ml) and water (32 ml). Potassium carbonate (3.65 g, 8.3equivalent) was added. After stirring for 15 hours at room temperature,the reaction mixture was heated up to 40° C. for 4 hours. The reactionmixture was concentrated under reduced pressure and the residue wastaken up in ethyl acetate (70 ml). Aqueous citric acid solution 15% (30ml) was slowly added followed by water (10 ml). The aqueous layer wasseparated and re-extracted with ethyl acetate (56 ml). The organicphases were washed with water (30 ml), 15% citric acid solution (40 ml)and saturated NaCl solution (30 ml). The organic layers were combined,dried over Na₂SP₄ and concentrated under reduced pressure to giveCyclosporine A diene (ISA_(TX)247) as a 98:2 E/Z mixture of double bondisomers (by 400 MHz NMR, ca 2–3% error). See R. W. Hoffmann, AngewandteChemie International Edition, Vol. 555 (1982); W. R. Roush,“Allylorganometallics,” Comprehensive Organic Synthesis, Pergamon Press,Vol. 2, pp. 1–53; and Y. Yamamoto, N. Asao, Chemical Reviews, p. 2307(1993).

Example 33 Preparation of Z-trimethyisilyl-protected Cyclosporine ADiene and Its Conversion to Z-Cyclosporine A Diene (ISA_(TX)247)

The trimethylsilyl-protected Cyclosporine A β-trimethylsilylalcohol (2g, 1 equivalent) was dissolved in THF (20 ml). The solution was cooledto 0–2° C. and potassium t-butoxide (4 equivalents) was added. After 1.5hours reaction, ethyl acetate (20 ml) and water (40 ml) were added. Theaqueous layer was separated and re-extracted with ethyl acetate (20 ml).The organic phases were washed with a saturated aqueous NaCl solution(20 ml). The combined organic phases were dried over Na₂SO₄, filtered,and concentrated under reduced pressure to give a mixture ofZ-trimethylsilyl-protected Cyclosporine A diene(trimethylsilyl-protected ISA_(TX)247), and Z-Cyclosporine A diene (theZ-isomer of ISA_(TX)247). The desilylation was completed by dissolvingthe crude product mixture in methanol (10% by weight in the solution)and adding a 1 M aqueous hydrochloric acid solution (1 equivalent).After 15 minutes at room temperature, water and ethyl acetate wereadded. The aqueous layer was separated and re-extracted with ethylacetate. The organic phases were washed with a saturated aqueous NaClsolution. The combined organic phases were dried over Na₂SO₄, filtered,and concentrated under reduced pressure, providing Cyclosporine A diene(ISA_(TX)247) as a 94:6 mixture of Z and E double bond isomers (by NMR).

Example 34 Preparation of E-Cyclosporine A Diene (ISA_(TX)247)

The trimethylsilyl-protected Cyclosporine A β-trimethylsilylalcohol (500mg, 1 equivalent) was dissolved in dichloromethane. This solution wascooled within a range of about 0–2° C., and treated withborontrifluoride diethylether complex (5 equivalents). After 1 hour,water (20 ml) and dichloromethane (20 ml) were added. The organic layerwas separated and washed with water (20 ml), dried over Na₂SO₄,filtered, and concentrated under reduced pressure to provide directlyCyclosporine A diene (ISA_(TX)247) as a 91:9 mixture by weight of the Eand Z double bond isomers (by NMR).

Example 35 Deprotection of Trimethylsilyl-protected Cyclosporine A Diene

Trimethylsilyl-protected Cyclosporine A diene was dissolved in methanol(10% by weight in the solution). This solution was treated with 1 Maqueous hydrochloric acid solution (1 equivalent). After 15 minutes atroom temperature, water and ethyl acetate were added. The aqueous layerwas separated and re-extracted with ethyl acetate. The organic phaseswere washed with a saturated aqueous NaCl solution. The combined organicphases were dried over Na₂SO₄, filtered and concentrated under reducedpressure, providing Cyclosporine A diene (ISA_(TX)247).

Example 36 Epoxidation of Acetyl Cyclosporin A

Acetyl cyclosporine A (2.0 g, 1.61 mmol) was dissolved in acetonitrile(30 mL). 1,3-Diacetoxy-acetone (0.14 g, 0.8 mmol) was added, followed by0.0004 M aqueous ethylenediaminetetra-acetic acid disodium salt (20 mL)and sodium bicarbonate (0.405 g, 4.82 mmol). To the stirred mixture,oxone (43.8% KHSO₅) (2.23 g, 6.43 mmol) was added portionwise over 2hours. The pH was maintained at 8.2 by constant addition of 1 N NaOH(total amount 6.4 mL) using a pH stat. The temperature was kept at22–25° C. by occasional cooling using a cold water bath. After 2.5 hoursthe reaction mixture was quenched by a few drops of a sodium bisulfitesolution. Water (100 mL) was added and the mixture was extracted twicewith tert-butyl methyl ether (100 mL, then 75 mL). The organic extractswere washed with dilute aqueous sodium chloride (100 mL), combined,dried over Na₂SO₄, and concentrated to afford crude acetyl cyclosporin Aepoxide (1.92 g, 95%; HPLC: 99.4% area) as a white solid foam.

Example 37 Preparation of Acetyl Cyclosporin A Aldehyde

Crude acetyl cyclosporin A epoxide (1.92 g, 1.52 mmol) was dissolved inacetonitrile (25 mL). Water (20 mL) was added, followed by sodiumperiodate (489 mg, 2.28 mmol) and 0.5 M sulfuric acid (3.05 mL, 1.52mmol). The reaction mixture was stirred at 40° C. for 18 hours, then theexcess sodium periodate was quenched by addition of aqueous sodiumbisulfite. Dilute aqueous sodium chloride (100 mL) was added and themixture was extracted twice with tert-butyl methyl ether (100 mL each).The organic extracts were washed with dilute aqueous sodium chloride(100 mL), combined, dried over Na₂SO₄, and concentrated to afford crudeacetyl cyclosporin A aldehyde (1.74 g, 92%; HPLC: 95.7% area) as a whitefoam. The crude product was chromatographed over silica gel using 40%acetone/60% hexane as eluent to give the product (1.41 g, 71% based onacetyl cyclosporin A; HPLC: 100% area) as a white solid foam.

Example 38 Preparation of Acetyl Cyclosporin A Aldehyde Using a One-potProcedure

Acetyl cyclosporin A (2.0 g, 1.61 mmol) was dissolved in acetonitrile(30 mL). 1,3-Diacetoxy-acetone (0.084 g, 0.48 mmol) was added, followedby 0.0004 M aqueous ethylenediaminetetra-acetic acid disodium salt (20mL) and sodium bicarbonate (0.405 g, 4.82 mmol). To the stirred mixture,oxone (43.8% KHSO₅) (1.67 g, 4.82 mmol) was added portionwise over 2hours. The pH was maintained at 8.2 by constant addition of 1 N NaOH(total amount 3.4 mL) using a pH stat. The temperature was kept at20–25° C. After 3.5 hours, 0.5 M sulfuric acid (5 mL, 2.5 mmol) wasadded to the reaction mixture, followed by a few drops of concentratedsulfuric acid, until pH 1.3 was reached. Then, sodium periodate (516 mg,2.41 mmol) was added, and the reaction mixture was stirred at roomtemperature for 2 hours and at 40° C. for 22 hours. Water (100 mL) wasadded and the mixture was extracted twice with tert-butyl methyl ether(100 mL, then 75 mL). The organic extracts were washed with diluteaqueous sodium chloride (100 mL), combined, dried over Na₂SO₄, andconcentrated to afford crude acetyl cyclosporin A aldehyde (1.9 g, 96%;HPLC: 83.4% area) as a white foam. The crude product was chromatographedover silica gel using 40% acetone/60% hexane as eluent to give theproduct (1.35 g, 68% based on acetyl cyclosporin A; HPLC: 100% area) asa white solid foam.

Example 39 (ISO) Wittig Reaction of Aceyl Cyclosporin A aldehyde with3-Dimethylaminopropyltriphenylphosphorylidene

A stereoselective synthesis of 1,3-dienes has been described by E. J.Corey and M. C. Desai in Tetrahedron Letters, Vol. 26, No. 47, pp.5747–8, (1985). This reference discloses that the ylide obtained bytreating 3-(dimethylamino)propyltriphenylphosphorane with potassiumhexamethyldisilazide can undergo a Wittig reaction with an aldehyde toform selectively a Z-alkenyldimethylamine. Oxidation of the amine withm-chloroperbenzoic acid gives the corresponding N-oxide which can thenbe heated in what is known as a Cope elimination to form the desired1,3-diene in which the configuration of the olefin formed during theWittig step is exclusively Z, or cis.

Analogously, the Z-isomer of ISA_(TX)247 may be prepared by reactingacetyl cyclosporin A aldehyde with ylide obtained by treating3-(dimethylamino)-propyltriphenylphosphonium bromide with potassiumhexamethyldisilazide. The resulting intermediate then undergoesoxidation, followed by Cope elimination to give acetyl-(Z)-ISA_(TX)247.Deprotection using a base results in (Z)-ISA_(TX)247. The oxidizingreagent may be metachlorperbenzoic acid.

To a stirred suspension of 3-dimethylaminopropyltriphosphonium bromide(2.5 g, 5.83 mmol) in anhydrous toluene (20 mL) was added potassiumhexamethyldisilazide (11.6 mL, 5.8 mmol, 0.5M solution in toluene)through a syringe. After stirring for 1 h at room temperature, thered-colored solution was centrifuged and the supernatant transferred toa reaction flask through a cannula. To the solid was added anhydroustoluene (10 mL), stirred and centrifuged. The supernatant wastransferred to the reaction flask and to the combined red-colored ylidewas added OAc-CsA-CHO (1.44 g, 1.17 mmol). Stirring was continued for afurther period of 2 h at room temperature when the color turnedlight-yellow. The reaction mixture was diluted with EtOAc (50 mL) andwashed subsequently with saturated NaHCO₃ solution (50 mL) and brine (50mL). Drying and solvent removal furnished a pale-yellow solid.Chromatography over a silica gel column and elution with acetone-hexanesmixture (gradient: 10 to 75% acetone and 90 to 25% hexanes) removed allphosphorous-related impurities. Further elution with acetone furnisheddesired product as a colorless solid (1.28 g, 84% yield). ¹H NMR (300MHz, CDCl₃): 2.23 (s, 6H), 2.03 (s, 3H), ¹³C NMR (300 MHz, CDCl₃):129.33, 126.95; MS m/z: 1301 (M⁺), 1324 (M+Na⁺).

Conversion to N-Oxide

To a stirred and cooled (0° C.) solution of the dimethylamino compoundobtained in the Wittig reaction (0.44 g, 0.34 mmol) in CHCl₃ (3 mL) wasadded a solution of m-CPBA (0.07 g, 0.405 mmol) in CHCl₃ (2 mL). Afterstirring for 30 min, dimethyl sulfide (0.5 mL) was added followed byCH₂Cl₂ (50 mL). Work-up by washing with NaHCO₃ solution (25 mL) andwater (25 mL), drying and solvent removal furnished a solid (0.43 g). ¹HNMR (300 MHz, CDCl₃): 3.19 (s, 3H), 3.18 (s, 3H), 2.03 (s, 3H). ¹³C NMR(300 MHz, CDCl₃): 131.89, 124.13; MS m/z: 1340 (M+Na⁺).

Cope Elimination of N-Oxide. Preparation of (Z)-Isomer of AcetylISA_(TX)247

The N-oxide (350 mg) was stirred neat and heated at 100° C. in vacuo for2 h. This was then passed through a column of silica gel. Elution withacetone-hexanes mixture (gradient, 5 to 25% acetone and 95 to 75%hexanes) furnished a colorless solid (314 mg). ¹H NMR (500 MHz, CDCl₃):6.49 (dt, J=16.99, 10.5 Hz, 1H); ¹³C NMR (400 MHz, CDCl₃): 132.20,131.09, 129.70, 116.85; MS m/z: 1279 (M+Na⁺).

(Z)-Isomer of ISA_(TX)247

To a solution of (Z)-acetyl ISA_(TX)247 (50 mg) in MeOH (4 mL) was addedwater (1.5 mL) and K₂CO₃ (60 mg) and stirred for 48 h at roomtemperature. The reaction mixture was stripped off solvents andextracted with EtOAc (20 mL). The organic layer was washed with water(10 mL) and brine (10 mL). Drying and solvent removal furnished acolorless solid. ¹H NMR (500 MHz, CDCl₃): 6.58 (dt, J=16.99, 10.5 Hz,1H); MS m/z: 1236.8 (M+Na⁺). The resulting compound was Z-isomer ofISA_(TX)247. No measurable E-isomer was observed by NMR.

Although only preferred embodiments of the invention are specificallydisclosed and described above, it will be appreciated that manymodifications and variations of the present invention are possible inlight of the above teachings and within the purview of the appendedclaims without departing from the spirit and intended scope of theinvention.

1. A method of preparing an isomeric mixture of cyclosporin A analogsmodified at the 1-amino acid residue, wherein the synthetic pathwaycomprises the steps of: a) protecting the β-alcohol of cyclosporin A byforming a first intermediate acetyl cyclosporin A; b) oxidizing theacetyl cyclosporin A to produce a second intermediate acetyl cyclosporinA aldehyde, wherein the oxidizing step is carried out with ozone and isan ozonolysis and further wherein the ozonolysis also comprisestreatment with a reducing agent; c) converting an intermediate acetylcyclosporin A aldehyde to a mixture of (E) and (Z)-isomers ofacetyl-1,3-diene by reacting the intermediate with a phosphorus ylidevia a Wittig reaction, optionally in the presence of a lithium halide;and d) preparing a mixture of (E) and (Z)-isomers of cyclosporin Aanalogs modified at the 1-amino acid residue by treating the mixtureof(E) and (Z)-isomers of acetyl-1,3-diene with a base, wherein thereducing agent is tributyl phosphine.
 2. The method of claim 1, whereinthe ozonolysis is done at a temperature ranging from about −80° C. toabout 0° C.
 3. The method of claim 1, wherein a solvent used for theozonolysis is a lower alcohol.
 4. The method of claim 3, wherein thelower alcohol solvent is methanol.
 5. The method of claim 1, wherein thebase that is used to treat the acetyl-1,3-diene is selected from thegroup consisting of sodium hydroxide, sodium carbonate, potassiumcarbonate, sodium alkoxide, potassium alkoxide, NH₃, hydroxylamine,hydrazine, and lower dialkylamines.